Protocol for generation, quantification, and phenotyping of brain metastases in preclinical mouse models

Summary

The field of brain metastasis is rapidly expanding, yet no consensus exists on the most reliable quantification approach. We present a protocol for assessing metastatic burden in mice following intracarotid injection of tumor cells. We describe steps for surgical procedures, brain processing, cryo-sectioning, and slide preparation, followed by phenotypic characterization. We detail procedures for quantifying brain metastatic area using automated microscopy and semi-supervised image analysis.

For complete details on the use and execution of this protocol, please refer to Rodriguez-Baena et al.¹

Graphical abstract

Highlights

• •Protocol for generation and quantification of brain metastases in mouse models
• •Semi-automated imaging workflow using high-throughput microscopy and Fiji
• •Tissue integrity preservation for downstream phenotypic characterization
• •Enables comparative analysis of metastatic burden across experimental conditions

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

The field of brain metastasis is rapidly expanding, yet no consensus exists on the most reliable quantification approach. We present a protocol for assessing metastatic burden in mice following intracarotid injection of tumor cells. We describe steps for surgical procedures, brain processing, cryo-sectioning, and slide preparation, followed by phenotypic characterization. We detail procedures for quantifying brain metastatic area using automated microscopy and semi-supervised image analysis.

Before you begin

The following protocol outlines the detailed steps for the accurate quantification and characterization of tumor burden in experimental brain metastasis mouse models. The use of fast quantification methods can lead to inaccurate results which could lead to misinterpretation of scientific results. Bioluminescence is a method that yields interesting results for in-vivo analysis by using luciferase reporters which are typically engineered into tumor cells. However, the results do not always match real tissue metastatic burden (Figures 1A and 1B). Also, fluorescence bulk quantification methods do not match the sensitivity required to detect small and big metastatic foci (Figure 1C). Here, we describe a protocol to generate mouse brain metastases, coupled with an accurate methodology to quantify the metastatic burden. For this, we employ intracarotid injections.² The main advantage of this technique is the production of experimental brain metastases with high reproducibility and lower variability. To improve the procedure we also incorporated the closure of the external carotid artery as described by Liu & colleagues.³ This protocol covers the complete workflow. This includes subsequent brain processing, vibratome sectioning, slide preparation, semi-automated image analysis, and manual quantification using Fiji/ImageJ (Figure 1D). This methodology is optimized for high-throughput microscopy and enables further phenotypic characterization of the tumor microenvironment from the same samples. The protocol has been successfully applied to evaluate the impact of immune modulation in brain metastases using NRAS∗ and B16F10 mouse melanoma models,^1,4 and can be adapted for other brain metastasis models or tumor types. For reproducibility and serving as an example we have also included an in-house developed non-pigmented melanoma cell line named FCT2 with tdTomato endogenous fluorescence.

Figure 1.

Brain metastasis burden quantification methods

(A) Bioluminescent signal detected by IVIS in mice with brain metastases established upon injection of luciferase expressing melanoma cells.

(B) Pictures of brains from mice in Figure 1A.

(C) Image of a mouse brain with metastases generated upon injection of tdTomato expressing FCT melanoma cells.

(D) Schematic representation of the process followed to generate brain metastases, sample processing and quantification of the metastatic burden.

A complete list of reagents, materials, and equipment is included in the key resources table. Detailed instructions for solution preparation, equipment calibration, and software setup are provided in the materials and equipment section.

Institutional permissions

All experiments involving animals were performed in accordance with the European Community Council Directive (2010/63/EU) and Spanish legislation. All experimental procedures described in this protocol were approved by CSIC and UMH Ethical Committees and the Animal Welfare Committee at the Instituto de Neurociencias CSIC-UMH. Since this protocol involves use and sacrifice of live vertebrates, any readers performing this protocol will need to acquire approvals from the relevant institutions.

Cell culture preparation

Timing: 1–2 h

• 1.In the days prior to tumor cell injection, plate the required number of cells to ensure sufficient yield for the planned number of animals.
• 2.Prepare at least three times the number of cells needed for injection to account for potential loss during processing (e.g., syringe loading).

Note: For example, if injecting 1 × 10⁵ cells per animal for 10 animals, prepare at least 3 × 10⁶ cells.

CRITICAL: Perform pilot studies to determine the optimal confluence state, injection cell number, and culture conditions for each specific cell line and experimental goal.

Pre-surgery preparations

Timing: 1–2 h

• 3.
  Sterilize all surgical instruments the day before tumor cell injection using autoclaving or gamma irradiation. The minimum required instruments include (Figure 2A):

  • a.
    2× No. 5 Dumont forceps.
  • b.
    1× Narrow serrated forceps.
  • c.
    1× Guthrie retractor or a similar tool to retract the tissue.
  • d.
    1× Fine scissors.
• 4.
  Prepare the animals for surgery by trimming and waxing the neck area.

  • a.
    Anesthetize animals using an isoflurane induction chamber.

    • i.
      Induce anesthesia with 3–4% isoflurane in an induction chamber until the animal is non-responsive to gentle toe pinch.
    • ii.
      During surgery, anesthesia is maintained at 1.5–2% isoflurane via a nose cone.
    • iii.
      Always keep monitoring anesthesia depth via using reflex responses such as toe pinch reflex, palpebral reflex (gentle touch near the eye) and breathing rate (60–90 breaths per minute).
  • b.
    Expose the ventral neck area and trim the hair using an appropriate animal hair clipper.
  • c.
    Apply depilatory wax to remove residual hair and keep the animal in the chamber for one additional minute.
  • d.
    Wipe the area with a cloth soaked in 70% ethanol. Ensure complete removal of residue to avoid skin damage. Dry the area with a clean cloth and return the animal to its housing cage.
• 5.Prepare buprenorphine at a concentration of 1 mg/mL and store at 4°C until use.

Figure 2.

Intracarotid surgery set up

(A) Surgery material indicated in section “materials and equipment”.

(B) Surgery reagents and supplies indicated in section “materials and equipment”.

(C) Complete picture of the surgery set up.

(D) Close-up picture of the surgery and stereomicroscope set up.

(E) Picture of the water bath.

(F) Picture of the heating plate for animal cages.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Primary antibody anti-mouse GFAP 1:1,000 working dilution	Merck	AB5541; RRID_AB177521
Primary antibody anti-mouse GFP 1:1,000 working dilution	Aveslab	GFP-1020; RRID_AB10000240
Primary antibody anti-mouse P2RY12 – 1:400 working dilution	Anaspec	AS-55043A
Primary antibody anti-mouse KI67 – 1:500 working dilution	Abcam	AS-55043A; RRID_AB2298886
Primary antibody anti-mouse TMEM119 – 1:400 working dilution	SySy	400 004; RRID_ AB_2744645
Secondary antibody anti chicken Alexa Fluor 488– 1:1,000 working dilution	Thermo Fisher Scientific	A11039; RRID_AB2534096
Secondary antibody anti-rabbit Alexa Fluor 488 – 1:1,000 working dilution	Thermo Fisher Scientific	A11008; RRID_AB143165
Secondary antibody anti-rabbit Alexa Fluor 568 – 1:1,000 working dilution	Thermo Fisher Scientific	A11011; RRID_AB143157
Secondary antibody anti guinea pig Alexa Fluor 568 – 1:1,000 working dilution	Thermo Fisher Scientific	A11075; RRID_AB141954
Secondary antibody anti guinea pig Alexa Fluor 647 – 1:1,000 working dilution	Thermo Fisher Scientific	A21450; RRID_AB2535867
Secondary antibody anti-rabbit Alexa Fluor 647 – 1:1,000 working dilution	Thermo Fisher Scientific	A31573; RRID_AB2536183
Secondary antibody anti chicken Alexa Fluor 647– 1:1,000 working dilution	Thermo Fisher Scientific	A32933; RRID_AB2762845
Chemicals, peptides, and recombinant proteins
Isoflutek 100 mg/g	Karizoo	CN586259.0
Ketamine hydrochloride, Ketamidor 100 mg/mL	Alivira	CN580395.1
Xylazine hydrochloride, Xylasol 20 mg/mL	Alivira	CN579700.7
Bupredine, 0.3 mg/mL	Dechra	CN 585314.7
Sucrose	PanReac	141621.1211
Paraformaldehyde	Sigma	104005
Mowiol	EMD Millipore	475904-100mg
Dabco	Sigma	D27802
Ethylene glycol	Scharlab	ET01641000
Glycerol	PanReac	141339.1211
10× PBS	Gibco	14200–075
ddH2O	Gibco	15230–147
Tris-HCl	Sigma	T15760
NaN3 (3 mM)	Sigma	71289-5g
Hans F-12 medium	Biowest	L0135-500
Fetal bovine serum	Capricorn	FBS-11A
Pen/Strep	Biowest	L0022-100
Saline solution	Braun	CN 570950
OCT	Sakura	4583
BSA	Sigma	A8022-100g
Tween 20	Sigma	P9416-50ML
DAPI	Sigma	D9542-10mg
Ophthalmic gel for mice (Recugel Gel Oftálmico)	Welnia	0023–9205
Deposited data
Fiji Macro	This paper	https://github.com/scijrb/Macro_Brain_Area
Experimental models: Cell lines
1014 Nras∗ cell line	Prof Lionel Larue	INSERM, France
FCT2 mouse melanoma cell line	In-house developed	--
B16F10 mouse melanoma cell line	ATCC	CRL-6475
Experimental models: Organisms/strains
Mouse: B6.129P2(Cg)-Cx3cr1tm2.1(cre/ERT2)Litt/WganJ (8-10 weeks old; males and females)	Referred in paper as Cx3cr1	RRID:IMSR_JAX:021160
Software and algorithms
Fiji	NIH, USA	ImageJ 1.54f
Zen blue	Zeiss	v3.8
Biorender	BioRender, Inc.	–
Others
Eppendorf centrifuge 5810R	Eppendorf	5810000010
Stereomicroscope Leica MZ6	Leica	MZ6
Cryotome	Microm	HM-450
Axioscan Z1	Zeiss	430038–90
Light source	SCHOTT	KL 1600 LED Light Source
Heating pad	UFESA	Flexy Heat N
Water bath	Selecta	JP Selecta 6001195
Superfrost slides	Epredia	HL26766
Falcon 15 mL conical centrifuge	Corning	CLS352096
Forceps Dumont 55	FST	11255–20
Forceps standard pattern	FST	11002–12
Fine scissors ToughCut	FST	14058–09
Retractor	FST	17021–13
Autoclip system	FST	12020–00
Microsurgical needles with suture thread	FST	12052–08
Peristaltic pump	Cytiva	18-1110-91

Materials and equipment

Specific equipment and materials are listed in the key resources table.

Note: Validation must be performed if reagents are used from other suppliers. Alternative equipment with similar characteristics as those described in the KRT can be used. For example, centrifuges capable of operating at 300 × g can be used for cell centrifugation, while cryotomes, cryostats, or vibratomes suitable for sectioning tissues at 100 μm are appropriate for quantifying metastatic burden. For imaging, both automated platforms and manual microscopy are valid alternatives.

Surgery set up

This is the recommended surgical material for the optimal process (Figures 2B–2F).

• •Stereomicroscope.
• •Light source.
• •Ophthalmic gel.
• •Surgery clips.
• •Sutures.
• •Sterile cotton.
• •Cotton swabs.
• •Sterile gauze.
• •Immobilization glass and rubber bands.
• •Heating pad.
• •Water bath at 37ºC.
• •1 mL syringes.
• •30G needles.
• •Isoflurane.
• •Buprenorphine.
• •Saline solution.
• •Povidone Iodine solution.

Recipe for freezing medium

• •In a 1 L measuring cylinder add the reagents and mix well. Transfer the mixture to a 1 L glass bottle and keep it refrigerated.

Reagent	Final concentration	Amount (for 1 L)
Ethylene Glycol	30%	300 mL
Glycerol	30%	300 mL
10× PBS	1×	100 mL
ddH2O	1×	300 mL
Total	–	1000 mL

Note: This recipe should be stored at 4ºC and can be used up to two months since preparation. Please note that the reagent consistency is viscous, and it should be handled with care.

Recipe for Mowiol solution

• •Use a 50 mL falcon tube and add the following reagents with the order stated below.

Reagent	Final concentration	Amount
Glycerol		6 grams
Mowiol		2.4 grams
ddH2O		6 mL
0.2 M Tris-HCl pH 8.5 (0.2 M)		12 mL
NaN3 (3 mM)	0.02%	4 μL
Dabco	2.5% (w/v)	0,5 grams

• •Add 6 g glycerol and a stir bar to a 50-mL plastic tube.
• •Add 2.4 g Mowiol (Calbiochem) and stir.
• •While stirring, add 6 mL ddH₂O and incubate for 2 h at RT (22°C–25°C).
• •Add 12 mL 0.2 M Tris-HCl (pH 8.5).
• •Add sodium azide to a final concentration of 0.02%.
• •Incubate at 50°C–60°C for 10 min or leave overnight (14–16 h) at RT (22°C–25°C).
• •Add DABCO to a final concentration of 2.5% (w/v) and vortex until fully dissolved.
• •Centrifuge at 5000 × g for 15 min. Aliquot 1 mL per tube and store at −20°C.
• •Before use, bring tubes to RT (22°C–25°C). Opened tubes can be stored at 4°C for up to 1 month. Discard if crystallization occurs.

Note: Use 15–20 μL for 22 × 22 mm coverslips and 40–50 μL for 22 × 50 mm coverslips. Avoid excess volume to prevent section displacement.

Recipe for 30% sucrose (1 L)

• •Weigh 300 g sucrose in a 2-L beaker.
• •Add 500 mL ddH₂O and stir until fully dissolved.
• •Transfer to a 1-L cylinder and top up with ddH₂O to 1 L.
• •Store in a glass bottle or aliquot in 50-mL tubes and freeze.

Note: For long-term storage, filter the solution in a sterile hood and store in sterile tubes. Unfiltered solution should not be used beyond 3 weeks due to risk of contamination.

Recipe for 4% paraformaldehyde (1 L)

• •Add 800 mL 1× PBS to a 1-L glass bottle.
• •Add 40 g PFA powder and stir at 60°C in a fume hood or water bath.
• •To aid dissolution, add 5N NaOH dropwise until the solution clears.
• •Cool to RT (22°C–25°C), filter to remove particulates, and top up with PBS to 1 L.
• •Adjust to pH 7 with NaOH or HCl if needed.
• •Aliquot and freeze until use.

CRITICAL: PFA releases toxic formaldehyde vapors. Always work in a fume hood and wear appropriate PPE including a face mask.

Note: PFA powder does not dissolve instantly, you need to raise the pH of the mixture by adding 5N NaOH drop by drop until a clear solution is formed. There may be small undissolved particles. Cool the solution to RT (22°C–25°C) and filter to remove particles.

Recipe for blocking solution and antibody solution

• •Blocking Solution: 3% BSA, 1% Normal Goat Serum (NGS), and 0.05% Tween-20 in 1× PBS.
• •Antibody Solution (Primary and Secondary): 1% BSA and 1% NGS in 1× PBS.

Step-by-step method details

Surgery and tumor cell injection

Timing: approx. 2–3 h for a group of 7–10 animals; 15–20 min per mouse surgery

This section outlines how to obtain and prepare tumor cells for carotid artery injection in mice and describes the complete surgical procedure. In this case, the experiment example has been executed with the mouse melanoma cell line NRAS 1014∗ which grow in complete F12 medium.⁴ To improve efficiency, consider teamwork: one person can prepare the tumor cell suspension while another sets up the surgery room and prepares the animals.

Note: In addition to adhering to institutional and national and international ethical guidelines, when possible, animals are randomly allocated to experimental groups using computer-generated randomization. For experimental groups with different genotypes, randomization is not possible as group assignment is determined by genotype. To reduce bias, experimental groups are composed to ensure equal representation of variables such as age and sex. Outcome assessments, including quantification of metastatic burden, are performed blinded to treatment conditions to reduce bias and improve reproducibility. It is important to adhere to these practices to keep up the maximum astringency level while performing experiments to ensure results reliability.

• 1.
  Prepare the surgical setup before harvesting the cells.

  • a.
    Set up the stereomicroscope area and disinfect all surfaces with 70% ethanol. Adjust the illumination for optimal surgical visibility.
  • b.
    Arrange all surgical materials, but keep the sterile instruments sealed until the surgery begins.
  • c.
    Prepare the anesthesia system, heating pad, and surgical table. Place animals in the induction room or area.
• 2.
  Harvest and prepare the tumor cells.

  • a.
    Add 1–2 mL of 1× trypsin (for 10 cm dishes; adjust accordingly) and incubate at 37°C for 2–3 min.
  • b.
    Add complete DMEM with FBS and pipette to create a single-cell suspension.
  • c.
    Transfer the suspension into a Falcon tube.
  • d.
    Centrifuge at 300 × g for 5 min at RT (22°C–25°C).
  • e.
    Aspirate the supernatant, resuspend the pellet in 1× PBS, and centrifuge again.
  • f.
    Resuspend the final pellet in 1 mL PBS. Pipette to homogenize the pellet using a 1 mL filter tip.
  • g.
    Count cells using a Neubauer chamber or an automated cell counter.
  • h.
    Prepare aliquots with the desired concentration.

    Note: In this article we use Nras∗ 1014 melanoma cells to a concentration of 2×10⁵ cells in 100 μL PBS per mouse.

  • i.
    Keep tubes on ice and transport them to the surgery room.

    Note: Depending on the number of animals to be injected, it may be necessary to harvest cells in batches. While keeping cells on ice helps preserve viability over extended periods, this step should be optimized for each cell line. It is recommended to evenly distribute mice from different experimental groups across the batches. Additionally, leftover tumor cells from each batch should be replated after surgery and confirm viability the following day.

    Note: A pilot experiment with cell titration must be performed to know the optimum cell number for intracarotid injections.

• 3.Cut small pieces of saline-wet cotton, approximately 4–5 mm² and keep them over a sterile surface.
• 4.
  Place the first mouse in the isoflurane induction chamber and wait until no signs of reflects are present.

  • a.
    Place the mouse under the stereomicroscope set-up attached to the air-flow isoflurane application. Proceed when no reflexes are observed.
  • b.
    Apply the ophthalmic gel solution.
  • c.
    Inject the Buprenorphine solution subcutaneously previously calculated by mouse weight.
  • d.
    Position the animal ventral side up, securing limbs under rubber bands over a heated glass pad.
  • e.
    Clean the depilated area with a gauze with povidone-iodide or 70% EtOH and let it dry.
  • f.
    Adjust the proper focus under the stereomicroscope and open the surgical tool sterile bag.
  • g.
    Place two #55 forceps to your left- and right-hand positions. Scissors and serrated forceps to the right (adjust these instructions for right or left-handed personnel).
• 5.Use serrated forceps and scissors to make a skin incision just left of the trachea (Methods Video S1).

Note: The whole surgery with instructions has been recorded and explained in the Methods Video S1.

• 6.Switch to forceps #55 and retract adipose tissue until the neck muscle is exposed. Use a retractor to secure fat tissue laterally.
• 7.Use both forceps to gently break down connective tissue and locate the carotid artery.

Note: Carotid artery appears opaque red, distinguishing it from adjacent vessels.

• 8.Carefully pass the tip of the forceps beneath the artery to detach it from underlying tissue. Confirm its elasticity but avoid excessive manipulation.

CRITICAL: Avoid touching the aorta, located beneath the fat tissue—damage is fatal. Do not manipulate the vagus nerve, recognizable by its white/gray rings, as it regulates gastrointestinal function (Methods Video S1).

• 9.
  Prepare the sutures:

  • a.
    Cut two sutures: one ∼4–5 cm, one ∼2 cm.
  • b.
    Place the short suture near the neck incision (close to the jaw).
  • c.
    Fold the long suture in half and coil it with gloved fingers. Pass the loop under the carotid and cut to produce two free threads. Unfold them into single threads.
  • d.
    Make a loose knot with both single threads. Pull from both knots and gently retract the artery to expose its Y-branch (Figure 1D and Methods Video S1).
  • e.
    Secure the external carotid artery by tying off one branch to prevent extracranial tumor seeding.
• 10.
  Isolate and prepare the injection site:

  • a.
    While pulling from both sutures, pierce the connective tissue of the Y artery connection and pass the short suture thread through it (Step 9b).
  • b.
    Make a firm knot and close the suture around the external carotid artery.

    Note: This will avoid tumor cells infiltrating in the extracranial regions of the jaw.

  • c.
    Release the sutures open knots and place one proximal and other distal from the carotid.
  • d.
    Take one of the saline-wet cotton pieces and place it beneath the carotid artery, leaving both suture open knots at both extremes. Accommodate the carotid artery by modifying the cotton depth and length so the blood flow is permanent (Methods Video S1).
  • e.
    Close the proximal knot (bottom) to the most southern point possible.

    Note: This will allow you to have as much carotid artery as possible to inject the tumor cells.

• 11.
  Tumor cell injection:

  • a.
    Load 100 μL of the cell suspension into the syringe. Remove all air bubbles as this could end up with the death of the animal.
  • b.
    Rest your hand on the surgical platform for stability. Do not hover or shake.
  • c.
    Angle the needle around 10° parallel to the carotid artery surface and try to inject as proximal as possible.

    Note: If you fail, you will have other attempts further up in the carotid artery.

  • d.
    Insert the needle bevel-up, ∼2/3 of the needle length into the artery.

    Note: You can help yourself by taking the left-hand forceps and pulling from the closed proximal suture. This will make the carotid less elastic and easier to pierce.

  • e.
    Press the plunger steadily and slowly until the whole content has been released. Do not release the needle or pull back yet.
  • f.
    With the left-hand forceps, pull up from the distal open knot so the circulation of the artery is closed. Then remove the needle and quickly close the distal suture by using both forceps.
• 12.
  Finish the surgery:

  • a.
    Remove the cotton and cut the excess of the suture threads.
  • b.
    Rinse with saline solution to clean the wound and close it with staples.
• 13.
  Post-surgical care:

  • a.
    Remove the animal from the surgical set up and inject 0.5–1.0 mL of saline subcutaneously.
  • b.
    Place it back in its cage and onto the heating plate.

Note: The animal should be awake within the next 5 min, check for vital signs.

Methods Video S1. Internal carotid surgery and tumor cell injection detailed video, related to step 1

In this video a complete description of the internal carotid surgery and tumor cell injection to develop brain metastasis in mice are shown.

Continue monitoring mice post-surgery for signs of distress or complications. Follow institutional veterinary guidelines for analgesia and health checks.

See troubleshooting 1 for issues related with animal surgery.

Perfusion, tissue processing, sectioning, and mounting

Timing: 3 days from perfusion to sectioning and mounting

In this step, we describe how to process the brain tissue from cells-injected animals. This includes perfusion to obtain the best samples for downstream analysis such as metastatic burden quantification and phenotypic characterization. We also detail the cryoprotection procedure to preserve tissue integrity, followed by sectioning and mounting.

Depending on the tumor cell line, animals may be sacrificed within 10–30 days post-injection and brains collected for analysis. Pilot experiments to establish experimental parameters, such as the expected time window for metastasis development, the onset of clinical progression, and the number of animals needed to achieve sufficient statistical power for downstream analyses should be performed. Always follow institutional and national ethical guidelines.

Note: Perfusion varies depending on multiple factors such as pump function, tubbing, labware and temperature. In the example presented in this protocol, the peristaltic pump used was, a Cytiva 18-1110-91 with 2.1 mm i.d. tubing in the ×1 range. The dial was set to position 3 which get an approximate rate of 1 mL/min. Please calibrate the instrument according to manufacturer's instructions.

Note: Successful tissue perfusion can be readily assessed by observing the clearing of the lungs and liver. Additionally, the efflux from the right atrium should appear clear with no signs of blood. For the brain, which will be perceived later, the color should also appear white in contrast to a reddish-cream-like color of a non-perfused sample.

• 14.
  Prepare the perfusion setup according to the number of animals. Gather the following materials:

  • a.
    Perfusion bomb and tubing.
  • b.
    Cold 4% PFA.
  • c.
    Cold 1× PBS.
  • d.
    Cold 30% sucrose.
  • e.
    25g needles.
  • f.
    Anesthesia.
• 15.
  Anesthetize animals via intraperitoneal injection of ketamine/xylazine and place them on a perfusion tray.

  • a.
    Open the rib cage, cut the atrium, and pierce the right ventricle. Perfuse with cold PBS for ∼2 min.
  • b.
    Switch to cold 4% PFA and perfuse with ∼35 mL (20–25 min).
  • c.
    Dissect out the brain and transfer it to a 50 mL tube containing 8–10× its volume of 4% PFA. Fix overnight (14–16 h) on a rotator.

Note: Check on the perfusion set up and instruments. Perfusion speed varies among protocols and instruments.

Pause point: Tissue fixation overnight (14–16 h).

Methods Video S2. Cryotome sample processing, relate to step 2

This video shows the complete process of cryotome sample preparation for metastatic burden quantification and sample phenotyping.

See troubleshooting 2 for issues related with fluorescence background with perfusion.

• 16.
  Wash out the PFA from the tissue:

  • a.
    The next morning discard the PFA and wash 3 times with cold PBS for 10 min each.
  • b.
    In the last wash, add 15% of cold sucrose solution and leave it at 4ºC until the tissue sinks to the bottom of the tube.

    Pause point: This step typically takes around 4–6 h. Is better if performed early in the morning.

  • c.
    Then change it to 30% sucrose and leave it overnight (14–16 h).

    Pause point: Overnight (14–16 h) step.

    Note: The next morning the brain should be at the bottom of the tube, indicating that is ready for cryo-sectioning.

• 17.
  Cryo-sectioning of the brain metastasis samples (Figure 3).

  Note: The complete process of cryotome sample processing has been recorded and edited in video (Methods Video S2).

  • a.
    Place the 30% sucrose tubes in an ice box and take the freezing media to the cryotome.
  • b.
    Set the cryostat to −20°C. Once ready, embed the brain in OCT with the cerebellum facing down. Freeze completely prior starting the sectioning.

    • i.
      For analysis of metastatic burden: cut 100 μm sections.
    • ii.
      For phenotypic analysis: cut 20–25 μm sections.
    • iii.
      Alternate cuts (e.g., 1 × 100 μm followed by 4 × 25 μm) to balance sample usage. This way we dedicate the same amount of material to both applications.
  • c.
    Place each section into a well plate with freezing medium using a fine brush. Label to distinguish between samples to assess metastatic burden and the sections for phenotyping.
  • d.
    Expect ∼36–40 sections (100 μm) and ∼140–180 sections (25 μm) per adult brain.

    Note: Some cryotomes allow for semi-automatic alternating section thickness, facilitating the workload.

    Pause point: You can place the sections at −20ºC and continue later.

• 18.Keep the phenotyping sections at −20ºC and proceed immediately with the mounting of the sections to quantify metastatic burden.
• 19.
  Mounting brain sections for imaging. Allow frozen tissue to equilibrate to RT (22°C–25°C) for 10–15 min. Proceed based on imaging type. For a detailed section mounting set up check Figure 4A.

  • a.
    Mounting for bright-field microscopy:

    Note: In this case, tumor cells like melanoma, which produce melanin pigment, can be easily identified with bright field microscopy and no additional biochemical staining is needed.

    • i.
      Take the content of one well which contains all the section of a single mouse brain and place them in RT (22°C–25°C) 1× PBS.
    • ii.
      Take the slices one by one and place them onto 2 glass slides and let them dry over a filter paper facing up.
    • iii.
      Add ∼200 μL Mowiol (without anti-fade), and an appropriate coverslip. Dry overnight, protected from light. (Figure 4B).
  • b.
    Mounting for fluorescence microscopy.

    Note: When tumor cells have a fluorescent reporter.

    • i.
      Prior to step 19.a, take the tissues from the freezing media to another well plate with PBS and DAPI and incubate for 30 min protected from light.
    • ii.
      Continue the protocol from step 19.a onwards. In this case, use Mowiol with anti-fade reagent to protect fluorescence from photobleaching. (Figure 4C).

      Pause point: Overnight (14–16 h) drying step.

      Note: The quality of the tissue will be clearly visible when mounting the tissues onto the glass slides. Tissues tearing apart will indicate poor quality tissue and should be discarded.

      Note: Excess of Mowiol when mounting the samples will affect the glass appearance and will require an extra cleaning step prior to the next steps.

      Note: To ensure data quality and reproducibility, only sections meeting specific criteria are used for analysis. Sections must be intact, flat, and with a uniform thickness of 100 μm Samples should be excluded if more than 10% of sections are damaged. All sections need to undergo visual inspection under bright field to confirm integrity before imaging.

      See troubleshooting 2 for issues related with Mowiol and bubble appearance.

Figure 3.

Cryotome sample processing

(A) Cryotome set up with necessary materials. OCT wax, forceps, brain tissue and cryosectioning device.

(B) Close-up picture of the cryotome with a frozen brain sample.

Figure 4.

Sample mounting

(A) Picture of the set up to mount the brain slices.

(B) Picture of a slide containing brain sections for metastatic burden assessment after intracarotid injection of the melanoma pigmented Nras∗ 1014 cell line.

(C) Picture of a slide containing brain sections for metastatic burden assessment after intracarotid injection of the melanoma non-pigmented FCT cell line.

Tissue imaging

Timing: 1 or 2 days to imaging

This section details the process of image acquisition for the quantification of metastatic burden and the phenotypic analysis. Imaging is performed using a semi-automated platform that enables high-throughput and reliable sample processing. Although this step can be time-consuming, it can be overlapped with free-floating immunofluorescence phenotyping (see Section Sample phenotyping characterization or point 26).

• 20.Load slides into the Axioscan Z1 microscope imaging platform. This system accommodates up to 4 slides per tray, with a total of 50 trays (200 slides per scanning round), allowing imaging of approximately 100 animals per session.
• 21.
  For light microscopy imaging:

  • a.
    Create a new profile and select the following criteria:

    • i.
      Select Whole Slide Acquisition using a 5× low magnification objective in bright-field mode. Tissue recognition is not required.
    • ii.
      Set Coarse Focus using the “onion layer” method with a 0.5 parameter. Fine focusing is unnecessary at this magnification.
    • iii.
      Assign individual file names to each slide for traceability.
    • iv.
      Estimated imaging time: ∼7 min per slide.
• 22.
  For fluorescence microscopy imaging:

  • a.
    Create a new profile and select the following criteria:

    • i.
      Select Whole Slide Acquisition using a 10× objective. Tissue recognition is not required.
    • ii.
      Set Coarse Focus using DAPI at 5× magnification with the onion layer method (0.5 parameter).
    • iii.
      Set Fine Focus using DAPI at 10×, selecting every Nth tile with a fine focus parameter of 3.
    • iv.
      Choose the desired fluorescence channels based on your sample (e.g., DAPI only or tumor-intrinsic fluorescence)
    • v.
      Assign individual file names to each slide.
    • vi.
      Estimated imaging time: ∼30–40 min per slide.

See troubleshooting 3 for issues related fluorescence background with image acquisition.

• 23.
  Exporting images:

  • a.
    Quick Export for bright-field imaging. This is available in the left image panel.
  • b.
    Batch Export for fluorescence imaging. This is available through image processing and export tools.

Note: Imaging platforms are typically shared in microscopy core facilities and may be subject to scheduling limitations. To optimize workflow, begin free-floating immunofluorescence-based phenotyping while imaging is ongoing, as this also requires 2–3 days.

Alternatives: Other imaging systems may be used, provided they can generate high-resolution .tiff images sufficient to identify small metastatic lesions in the brain parenchyma. Optimize image quality to ensure reliable quantification in downstream analysis.

Metastasis burden analysis

Timing: from 1–8 h, depending on the number of animals per experiment. 10–20 min per animal

This section describes how to accurately quantify metastatic burden using the scanned images obtained from previous steps. Analysis is conducted using the open-source imaging software Fiji (or ImageJ as an alternative). The quantification process is divided into two main stages: First, quantification of the Total Brain Slice Surface Area. This step is semi-automated and utilizes a custom macro script to batch-process multiple images efficiently. It provides a reference area to normalize the metastatic burden in subsequent analyses. Quantification of the Metastatic Area: This step is performed manually. It involves identifying and measuring regions containing metastatic lesions based on image contrast or tumor-specific features (e.g., melanin pigmentation or fluorescence).

Note: Accurate quantification requires consistent imaging parameters and high-quality tissue preservation. Images with damaged or poorly preserved tissue should be excluded from analysis.

• 24.
  Image calibration and quantification of Brain Slice Area (common to both bright field and fluorescence):

  • a.
    Open a single .tiff image in Fiji/ImageJ. Identify the white cross on the superfrost slide—its horizontal bar measures 500 μm.

    • i.
      Go to Analyze → Set Scale, input the known distance (500 μm), and set it as global calibration (Figure 5A).
    • ii.
      This calibration will apply to all images during this session.
  • b.
    Open the Macro Recorder (Plugins → Macros → Record) and paste the following code. Save it as a .ijm file.

    Note: This macro will calculate the surface area of each brain slice:semi-automatically

    See troubleshooting 4 for issues related scale input.

    run("8-bit");

    run("Out [-]");

    waitForUser("Pause1", "Draw the crop area");

    run("Crop");

    run("Threshold...");

    run("Out [-]");

    waitForUser("Pause2", "Choose the appropriate threshold to exclude background");

    run("Convert to Mask");

    run("Analyze Particles...", "size=5316635.16-Infinity circularity=0.00-50.00 show=[Overlay Masks] display include summarize");

    selectWindow("Threshold");

    run("Close");
  • c.
    Execute the macro for each image. The macro will automatically prompt different messages with instructions:

    • i.
      Manually draw a crop around the brain section (Figure 5B).
    • ii.
      Set a threshold to exclude background noise and artifacts by sliding the bar to the left or right depending on the sample type (Bright field or fluorescence).

      Note: It will analyze the particles from one determined size to infinity to avoid small artifacts considering the circularity of the objects (Figure 5C).

    • iii.
      You may edit the macro with the following parameters:
    • iv.
      Auto-save particle analysis results.
    • v.
      Load next image in a batch from the same folder.
    • vi.
      Export overlays or summary tables automatically.
  • d.
    Do not close the results and summary windows until all images have been analyzed. When so, export the data or copy to a spreadsheet manager (Figure 5D).
• 25.
  Quantification of the Metastatic Area in bright field or fluorescence images:

  • a.
    Open a .tiff image in Fiji/ImageJ, ensuring that the global scale calibration is still active (see Step 23.a.1).
  • b.
    Use the Zoom Tool or +/− keys to focus on regions containing metastatic foci. In the case of bright field images, the metastatic foci will be visible thanks to the melanin accumulation (black spots). For the fluorescence images, the selection of DAPI and/or fluorescence reporters are necessary for its quantification.
  • c.
    Select the Freehand Selection Tool to manually outline each visible metastasis.
  • d.
    After outlining each focus, press T to add the Region of Interest (ROI) to the ROI Manager.
  • e.
    Repeat for all metastases in the image.
  • f.
    When all foci are selected press M to open the Results window containing measurement parameters: [Area], [Mean], [Min], [Max].
  • g.
    Save the ROI set by going to ROI Manager → More → Save..., saving as a .zip file for future reference. Name it with the sample name to keep track of the sample analysis.
  • h.
    Do not close the results and summary windows until all images have been analyzed. When so, export the data or copy to a spreadsheet manager.
  • i.
    Once finished, copy the information to an excel file.

Figure 5.

Metastasis quantification

(A) Image capture of the scale settings for quantification using Fiji.

(B) Step 1 of the Fiji Macro, selecting the desired area for quantification.

(C) Step 2 of the Fiji Macro, selecting the desired threshold for quantification.

(D) Threshold quantification of brain slices. Every element appears in an induvial color mask and represents a single brain section.

See troubleshooting 5 for issues related with thresholding inconsistencies and quantification.

See troubleshooting 6 for issues related no quantification data output.

• 26.
  Spreadsheet calculus:

  • a.
    Check that you obtain two values for each image:

    Note: Total Brain Area indicates the semi-automated quantification showing the total area of the brain sections. Metastatic area indicates results of the ROI quantification which will display the total area of the metastatic foci.

  • b.
    If instructions have been followed, you should have two slides per animal. First sum up the results of both datasets.
  • c.
    Calculate the % of metastatic are by getting the total area of Brain section and obtaining the % out of it.

    Note: Check the digits and numerical punctuation between your operative system and your spreadsheet manager. Fiji uses points (.) as thousand separators and this could lead to data misinterpretation.

    CRITICAL: Do not close Fiji/ImageJ during the quantification session. Closing the software will result in the loss of global scale calibration. If a break is necessary, keep the program open until all quantifications are completed.

    Note: Semi-automated quantification of metastases using threshold filtering and particle analysis in Fiji/ImageJ was evaluated but proved unreliable. Anatomical complexities in regions such as the thalamus, dentate gyrus, and cortex introduce signal artifacts and false positives. These limitations necessitate manual delineation for accurate quantification of metastatic foci.

    Note: To minimize variability in image quantification, a single trained user analyzes all experiments within a project. If multiple users are involved, independent quantifications are compared to ensure consistent trends across conditions. Compliance with standardized guidelines by all users ensures accurate and reproducible measurements of the metastatic area.

Sample phenotyping characterization

Timing: 2 days

This protocol benefits from the use of brains to perform the analysis of metastatic burden and phenotypic characterization. Thus, it allows to correlate metastatic burden and protein expression and spatial distribution to characterize cell states and behavior. We explain how to use the free-floating immunofluorescence staining technique to assess the biology of the brain metastasis samples at cellular level.

• 27.Choose the desired free floating brain sections and place them in a petri dish with 1× PBS to remove the excess of freezing media (Figure 4A).
• 28.
  Select an appropriate multi well plate depending on the number of slides to be processed simultaneously. It can be performed from 48 to 6 wells plates.

  • a.
    Place an appropriate volume of blocking medium (see recipes in the materials and equipment set up section) and using a brush place the sections in the medium. Incubate for 1 h at RT (22°C–25°C).
  • b.
    During the incubation time, prepare antibody medium (see recipe) and arrange the desired antibody combination.

    Note: In this case we have selected the following antibodies and its dilution:

    • Anti-mouse GFAP (1:1000).
    • Anti-mouse GFP (1:1000).
    • Anti-mouse P2RY12 (1:400).
    • Anti-mouse KI67 (1:500).
    • Anti-mouse TMEM119 (1:400).

    Note: Follow the manufacturer’s recommended antibody dilution or perform an in-house titration to know the appropriate volume. Inclusion of controls lacking the primary antibody is recommended to evaluate non-specific background signal from the secondary antibody. Additionally, testing multiple antibodies targeting the same antigen is advised to confirm specificity.

  • c.
    After the blocking, carefully aspirate the medium with a pipette and discard it.
  • d.
    Add the primary antibody solution and incubate sections protected from light at 4ºC overnight (14–16 h).

    Note: Depending on the laboratory set-up it would be interesting if the multi-well plate is incubated with a gentle shaking. This avoids tissue clumping and improves antibody distribution.

  • e.
    The next morning, wash 3× with 1× PBS 0.05% Tween 20. For this, aspirate the primary antibody solution and add the washing solution onto the well. Repeat three times and incubate 10 min each time in a rocker with gentle agitation.
  • f.
    Prepare the secondary antibody solution depending on the selected primary antibodies.

    Note: We have selected the following the secondary antibodies:

    • Alexa Fluor 568 antibody (1:1000).
    • Alexa Fluor 647 antibody (1:1000).

  • g.
    Additionally, add DAPI to a concentration of 1 μg/mL to the secondary antibody solution.
  • h.
    Incubate the mix protected from light at RT (22°C–25°C) and with gentle agitation for 1 h.
  • i.
    Repeat step 28e.
  • j.
    To mount the free-floating sections, use a 10 cm petri dish. Use the same set up as in step 27.
  • k.
    Insert a superfrost slide into the petri dish with PBS, leaving the white labeled side resting on the edge of the dish. In this way half of the plate is submerged at an angle of 15–20 degrees (Figure 4A).
  • l.
    Carefully drag the brain sections onto the glass using the brush. Once finished do not leave the section to dry for too long. Add the Mowiol with antifade reagent.

    Alternatives: Other commercial mounting media with anti-fade reagents can be used in this step.

  • m.
    Leave the section drying overnight (14–16 h) RT (22°C–25°C) protected from light.
• 29.The sections are ready for imaging. Multiple microscopy platform can be used to obtain fluorescence images and analyze protein expression and distribution.

Expected outcomes

This methodology enables accurate quantification of metastatic burden in the brain using a combination of semi-automated and manual procedures. These steps significantly expedite image processing while preserving tissue integrity, allowing for subsequent phenotypic characterization of the same specimens. By following this protocol, brain samples are sectioned and imaged using high-throughput microscopy, providing robust and reproducible measures of metastatic burden across experimental conditions.

We have extensively used this approach to measure and quantify the metastatic burden of NRAS∗ 1014 and B16F10 mouse melanoma cells. As described, we have employed genetical depletion of Rela to target the NF-kB pathway in Cx3cr1 expressing cells upon tamoxifen treatment (Rela cKO mice).¹ This approach allows us to compare the metastatic burden in two conditions, in this case, Rela WT and Rela cKO mice (Figure 6A). Compared to other existing methods—such as bioluminescence imaging, which can be affected by tissue opacity or signal quenching, or whole-brain clearing techniques, which preclude additional tissue analysis—our protocol offers precise and quantifiable results while retaining material for downstream applications. Additionally, this protocol can be extended to fluorescence application by directly imaging tumor cells with a reporter (Figures 6B and 6C).

Figure 6.

Expected results: Metastasis burden quantification

(A) Quantification of percentage of total brain metastatic area in an experiment performed injecting Nras∗ 1014 cells in vehicle or tamoxifen treated Cx3cr1YFPCreERT2-Rela^fl/fl mice. N = 9 animals per condition. Results are presented as mean ± SEM and differences between the two experimental groups were tested by Student’s t-test. ∗, p<0.05.

(B) Bright field image obtained by scanning a complete slide containing brain sections from mice injected with a pigmented melanoma cell lined along with a close-up picture for detail.

(C) Fluorescence image obtained by scanning a complete slide containing brain sections from mice injected with a non-pigmented TdTomato expressing melanoma cell line along with a close-up picture for detail.

Furthermore, this workflow facilitates the integration of immunophenotypic data, adding a valuable layer of insight into the metastatic microenvironment. Using this protocol, investigators can assess the spatial distribution and morphological changes of relevant cell populations. In our study, we employed well-established markers such as P2RY12 and TMEM119 for microglia, GFAP for astrocytes and KI67 to label dividing cells, to explore cellular dynamics in response to experimental treatments (Figure 7). This approach enables detailed investigations into cell-specific responses that accompany or modulate metastatic progression.

Figure 7.

Expected results: Sample phenotyping

(A) Fluorescence image of a brain metastasis edge depicting microglia stained with anti-GFP antibody (green) and anti-Ki67 (red) showing the nuclei of dividing cells. DAPI stains cell nuclei (blue).

(B) Fluorescence image of a brain metastasis edge depicting microglia stained with anti-P2RY12 antibody (green) and astrocytes labeled with anti-GFAP (red). DAPI stains cell nuclei (blue).

(C) Fluorescence image of a metastasis edge depicting astrocytes labeled with anti-GFAP (green) and microglia stained with anti-TMEM119 antibody (red). DAPI stains cell nuclei (blue).

(D) Fluorescence image of a metastasis edge depicting microglia stained with anti-TMEM119 antibody (green), dividing cell nuclei with anti-KI67 (red) and astrocytes labeled with anti-GFAP (magenta DAPI stains cell nuclei (blue). White bar scale = 20 μm.

Quantification and statistical analysis

Statistical analyses were performed using GraphPad Prism 8 software. For the example presented in this protocol (Figure 6A), results are presented as mean ± SEM and Student’s t-test was used to assess differences between the two experimental groups. Depending on the number of experimental groups, Student’s t-test or one-way ANOVA followed by Bonferroni’s multiple comparison test (for more than two groups) can be applied to assess statistical significance.

Sample size (e.g., N = 9 per condition in Figure 6A) was determined based on pilot experiments performed with the same model system to estimate variability in metastatic burden and anticipated effect size. These empirical observations guided our choice to use 8–10 animals per group to achieve an estimated statistical power of 80% (α = 0.05). No animals or data points were excluded from analysis unless explicitly stated.

All animals were randomized during surgery and data acquisition, and whenever feasible, analyses were performed blind to condition to reduce bias.

Limitations

While this protocol enables precise quantification of brain metastatic burden and facilitates downstream phenotypic analysis, it is not without limitations. One of the main constraints lies in the manual annotation for the quantification of metastatic foci. Although semi-automated approaches were tested using thresholding and particle analysis, they yielded inconsistent results due to the complex cytoarchitecture of brain regions such as the thalamus, dentate gyrus, and cortex, which often produced false positives. As a result, manual delineation remains necessary for accurate analysis, making this step time-consuming and potentially subject to inter-operator variability.

To mitigate such variability, all users are trained using standardized reference datasets, and once a user initiates the quantification of an experiment, they complete the entire dataset. When multiple users are involved, inter-rater reliability is assessed, and discrepancies are resolved through consensus.

Another limitation is the dependency on high throughput slide scanning platforms such as the Axioscan Z1. While this equipment significantly improves imaging capacity, access may be restricted in shared core facilities, leading to potential bottlenecks in image acquisition. Environmental factors such as fluctuations in ambient light or temperature are unlikely to affect the imaging directly, but scanner calibration and maintenance are critical for consistent results. In addition, tissue quality is essential; poorly fixed or processed brains can lead to sectioning artifacts or uneven staining, compromising both imaging and quantification accuracy.

Finally, while the protocol retains tissue for immunophenotyping, the quality of downstream immunofluorescence results can vary depending on antibody penetration, section thickness, and autofluorescence. These variables must be carefully controlled and optimized depending on the markers and imaging platforms used.

Troubleshooting

Problem 1

Problems during surgery (linked to step 1 to 12). During surgery some issue can arise which can be solved to minimize suffering and save the life of the animals.

• •Animal breathing changes (slower or faster).
• •Pinch in the carotid artery with forceps or scissors.
• •Introduction of air bubbles while tumor cell injection in the carotid artery.

Potential solution

The following solutions are proposed for the problems mentioned above.

• •Check the oxygen and isoflurane mixer to ensure the proper mix. Also check the tubbing system to inspect for possible leaks.
• •To avoid that the animal bleeds out, press the artery with a piece of cotton for at least 30 seconds. If the damage is small and bleeding controlled, surgery can be resumed after a few minutes.
• •Monitor the animal, finish the surgery, staple the wound and monitor it closely for the next 10–15 min for possible cardiac arrest. If animal seems unwell, sacrifice the mouse.

Problem 2

Bubbles in the Mowiol mounting medium interfere with image quantification (Linked to Step 18): If Mowiol is not at RT (22°C–25°C) when used to mount the slides for metastatic burden analysis, bubbles may form during application. Although the Fiji macro is designed to exclude these artifacts, delayed image acquisition may lead to bubble expansion or shrinking, potentially affecting automated quantification and requiring manual correction (Figures 8A–8C).

Figure 8.

Troubleshooting 2 and 3

(A) Picture of a good quality scanned slide.

(B) Picture of an acceptable quality scanned slide with bubbles within the Mowiol mounting medium.

(C) Picture of a non-acceptable quality scanned slide where Mowiol has shrink.

(D) Picture of a histogram from the software Zen Blue for the Axioscan Z1 controller showing the fluorescence and light histogram. The top panel shows a histogram where light information is scattered outside the histogram and therefore a bad example. The bottom picture represents a good example.

Potential solution

To avoid this problem.

• •Allow Mowiol to reach RT (22°C–25°C) for at least 30 min before use.
• •Use a Pasteur pipette instead of a pipette tip to minimize bubble formation during aspiration.
• •Acquire images as soon as possible after mounting. Over time, especially in 100 μm sections, Mowiol may shrink, increasing the chance of artifacts that interfere with analysis.

Problem 3

Autofluorescence in brain tissue interferes with signal interpretation (Linked to Step13 and 21). Endogenous autofluorescence from lipofuscin or blood remnants, especially in old or poorly perfused tissue. This causes problems and interference with image acquisition and metastasis quantification.

Potential solution

Perform appropriate perfusion with PBS before fixation to reduce blood content. Include autofluorescence quenching steps such as incubation with TrueBlack or Sudan Black B before immunostaining (steps not included in this protocol).

Alternatives: If autofluorescence is still problematic, consider using spectral unmixing or switching to chromogenic detection whenever possible.

Problem 4

ROI scale is lost when reopening images (Linked to Step 23 and Step 26). Closing Fiji/ImageJ resets global calibration, affecting all subsequent measurements.

Potential solution

Do not close Fiji/ImageJ until the full quantification session is completed. If a pause is needed, keep the software running in the background. If something fails (e.g. electricity shortcut etc.), make sure to annotate the pixel to distance ratio number when you set up the scale (Figure 5A).

Alternatives: As a precaution, save ROI sets and measurement tables frequently, and document calibration settings for re-application if needed.

Problem 5

Inconsistent thresholding during surface quantification using macro or no areas detected when running the macro (Linked to Step 25). The origin can be related to variable staining intensity or thickness cut or imaging conditions across slides. This problem will cause the thresholding selection to fail, and manual quantification should be performed.

Potential solution

Normalize imaging settings and illumination before acquisition. Use manual adjustment of thresholds within the macro as prompted, and test macro performance on representative images before batch processing. This will allow to acquire the best images possible for the image analysis. Always check the illumination setting diagram and make sure the light signal is contained within the histogram (Figure 8D). Make sure that the histogram selects the right signal and not background. When this happens, the red signal (you can modify this color at your will) should cover the brain sections (Figures 9A and 9B). If there are no signals after this modification you need to check the scale set up. It means that this step might have been skipped or the data misintroduced.

Figure 9.

Troubleshooting 4

(A) Picture of a Fiji Macro thresholding process showing a good detection of brain sections.

(B) Picture of a Fiji Macro thresholding process showing a wrong detection of brain sections due to opposite threshold selection.

(C) The top panel represents a picture of a Fiji Macro thresholding process showing an example of a non-acceptable quality image due to Mowiol shrinking problems. The bottom panel shows the results of manually deleting the background.

Also, this could be due to the bubbles in the mounting media. If the image acquisition was taken long after mounting, Mowiol shrinking problems may appear (Figure 8C), leading to inconsistent quantification results. Regrettably the only solution for this is to choose the freehand selection tool and delete the bubble area between the brain section and hit delete (Figure 9C). This would result in a manual background removal which has to be repeated manually until all the brain sections are well defined.

Alternatives: Modify macro parameters or apply pre-processing steps such as background subtraction for greater consistency.

Problem 6

Obtaining no data in metastatic quantification (Linked to step 24). After selecting the metastatic ROI when measuring the data the number of metastases is equal to zero or very small values. Normally this happens when the scale has not been set appropriately and the measurements are not calculated accordingly.

Potential solution

To fix this, open your image to calibrate the scale, measure that the (+) object does really measures 500 μm or the distance that you have chosen. Set up the global scale again and measure the metastatic ROIs again.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Berta Sanchez-Laorden (berta.lopez@umh.es).

Technical contact

Technical questions on executing this protocol should be directed to and will be answered by the technical contact, Francisco Javier Rodriguez-Baena (javier.rodriguez.baena@gmail.com).

Materials availability

This study did not generate new unique reagents.

Data and code availability

Original code generated during this study is available at Github: https://doi.org/10.5281/zenodo.15748814.

Acknowledgments

This work was supported by Ministerio de Ciencia, Innovación y Universidades, Agencia Estatal de Investigación (MICINN-AEI, PID2022-141613OB-I00 to B.S.-L.). We also acknowledge support from the GVA (CIPROM/ 2021/045) and Centro de Excelencia Severo Ochoa, grant CEX2021-001165-S, funded by MCIN/AEI/10.13039/501100011033. We thank Liu Z., Zhao J.J., and colleagues for the internal carotid injection image that has been adapted and was included in the graphical abstract and Figure 1A of the original manuscript¹ and in Figure 1D and the graphical abstract of this manuscript. This image was originally published in Sci. Rep. 9, 622³ under CC BY 4.0 (http://creativecommons.org/licenses/by/4.0/). Special thanks to laboratory members Alba Castillo and Pablo Ballesteros for their help and support. We thank the following facilities at the Instituto de Neurociencias CSIC-UMH that have facilitated the work: cell culture unit, omics facility, and the microscopy unit. Special thanks to the animal housing facility for their work and support. Illustrations were created with BioRender.

Author contributions

F.J.R.-B. conceived the study, designed and performed the experiments, and wrote the manuscript. B.S.-L. provided the funding, supervised the project, and revised the manuscript.

Declaration of interests

The authors declare no competing interests.