Immunohistochemistry and RNA in situ hybridization in mouse brain development

Abstract

During development, the mouse brain is progressively divided into functionally distinct compartments. Numerous neuronal and glial cell types are subsequently generated in response to various inductive signals. Each cell expresses a unique combination of genes encoding proteins from transcription factors to neurotransmitters that define its role in brain function. To understand these important and highly sophisticated processes, it is critical to accurately locate the various proteins and cells that produce them. In this chapter, we introduce the techniques of Immunohistochemistry, which detects the localization of specific proteins, and RNA in situ hybridization, which enables the visualization of specific mRNAs.

1. Introduction

The mouse brain consists of multiple divisions (cerebrum, epithalamus, thalamus, hypothalamus, cerebellum and brain stem), and more than 100 million neurons and glia [1]. Extrinsic inductive signals and intrinsic cellular programs both play key roles in the compartmentalization of the brain as well as cellular behaviors such as proliferation, differentiation, migration and cell death. To better understand the developmental processes involved in mouse brain development, it is important to obtain information regarding the spatial and temporal patterns of gene expression. In this chapter, we will describe methods for the detection of protein (immunohistochemistry, IHC) and mRNA (RNA in situ hybridization, RISH) in brain sections.

IHC detects particular proteins present in tissues. The principle underlying this technique is the specific bindings between antibodies and antigens. To visualize the antibody-antigen interaction, an antibody is tagged with a fluorophore, which can be conveniently detected with a fluorescent microscope. Alternatively, the antibody can be conjugated to an enzyme that catalyzes a color-producing reaction, which can be visualized under a regular microscope. In this chapter we will describe the method of IHC with the fluorophore-labeled antibody. The procedure comprises tissue preparation, blocking, primary and secondary antibody incubation, mounting and visualization.

The success of IHC heavily depends on the availability of high quality antibodies. In addition, secreted signaling proteins as well as proteins of the extracellular matrix are not restricted to the cells producing them, preventing a high resolution identification of the signaling centers. On the other hand, RISH allows the detection of the expression of virtually all genes in the cells, even genes that do not encode proteins, providing more flexibility compared with the antibody-based IHC method. Traditionally, RISH depends on the hybridization of the specific RNA sequence in situ to radiolabeled probes [2]. Currently, Digoxigenin-labeled probes are more commonly used in RISH, which can be recognized with antibodies coupled with fluorophore or enzymes such as alkaline phosphatase or peroxidase [3].

RISH can be performed on both frozen sections and paraffin sections, with frozen sections allowing more sensitive detection of weak signals [4]. The RISH method we introduce in this chapter uses Digoxigenin-labeled riboprobes (complementary RNA probes) to detect specific mRNA on frozen brain sections. The procedure includes synthesis of riboprobes, hybridization of sections with Digoxigenin-labeled riboprobes, post-hybridization washes, incubation with alkaline phosphatase (AP)-conjugated anti-Digoxigenin antibody, and a color reaction using the phosphatase substrate BM purple solution.

2. Materials

The materials should be stored at room temperature unless otherwise specified.

2.1. Materials for cryosection preparation

1. Dissection tools: student quality iris scissors (Fine Science Tools), Dumont forceps (Fine Science Tools), spoon (Fine Science Tools).

2. 6 cm petri dishes.

3. 24-well tissue culture plates (see Note 1).

4. Stereomicroscope, such as Nikon SMZ645.

5. Phosphate buffered saline (PBS): Prepare 10x stock by dissolving 80 g NaCl, 2 g KCl, 14.4 g Na₂HPO₄ and 2.4 g KH₂PO₄ in 1 liter of distilled, de-ionized water (ddH₂O). Adjust pH to 7.4 and autoclave at 121°C for 25 min. Dilute to 1x solution with ddH₂O for use.

6. 4% Paraformaldehyde (PFA): prepare 16% stock by adding 32 g PFA powder and 100 μl 5 N NaOH in 150 ml ddH₂O pre-warmed at ~65°C (see Note 2). Once PFA is dissolved, add 20 ml 10x PBS and adjust the volume to 200 ml with ddH₂O. Filter through Whatman paper and make 10 ml aliquot in 50 ml centrifuge tubes. Store at −20°C. Thaw one tube at ~65°C and dilute to 4% PFA with PBS before use. 4% PFA can be stored at 4°C for up to one week (see Note 3).

7. Nutator mixer (VWR).

8. 30% sucrose: Dissolve 12 g sucrose powder in 40 ml PBS. Store at 4°C.

9. Tissue-Tek® O.C.T. compound (VWR): Store at 4°C.

10. Disposable embedding molds.

11. Dry ice or liquid nitrogen in appropriate containers.

12. Cryostat, such as Leica CM1900.

13. Superfrost Plus microscopic slides (see Note 4).

14. Slide boxes.

15. Micro Slide trays.

2.2. Materials for immunohistochemistry

1. Coplin Jars.

2. Blocking buffer: 1% normal goat serum, 0.1% Triton X-100, in PBS. Keep at 4°C (see Note 5).

3. A humidified slide incubation chamber (Figure 1): Cut two 5 ml serological pipettes and tape them to the bottom of a flat-bottom plastic box with lid, such that the two pipettes are parallel and 5 cm apart. Place paper towels soaked with ddH₂O on the bottom to keep a moist environment.

4. Paper towels.

5. Fluorophore-conjugated secondary antibodies: Store at −20°C in 50% Glycerol.

6. Micro Slide trays.

7. Coverslips, 24×50 mm.

8. Forceps: Dumont forceps (Fine Science Tools).

9. Dabco® 33-LV: Aldrich. Store at 4°C (see Note 6).

10. Nail polish.

Figure 1.

A home-made humidified slide incubation chamber. This chamber is made by taping two 5 ml serological pipettes, broken to appropriate length, to the bottom of a plastic container with lid. Paper towels soaked in ddH₂O provide humidity during the incubation. For RISH, a microwavable box or a container that can withstand 55°C temperature is needed.

2.3. Materials for the synthesis of RNA probes

1. A plasmid with the promoters for viral RNA polymerases (T3, T7 and Sp6) flanking the multiple cloning sites, Store at −20°C.

2. Restriction endonucleases: Store at −20°C.

3. Horizontal DNA electrophoresis apparatus and power supply.

4. Agarose.

5. 10 mg/ml Ethidium Bromide solution: keep away from light (see Note 7).

6. TBE buffer: Dissolve 54 g Tris base, 27.5 g Boric Acid in ddH₂O, add 20 ml 0.5 M EDTA (PH 8.0), bring the volume to 5 liters with ddH₂O.

7. 1 kb DNA ladder.

8. 3 M sodium acetate (NaOAc), pH 5.2: Dissolve 408.3 g of NaOAc•3H₂O in 800 ml ddH₂O. Adjust pH to 5.2 with acetic acid and adjust the volume to 1 liter with ddH₂O. Aliquot and autoclave.

9. Phenol saturated with Tris•HCl, pH8.0: Store at 4°C.

10. Chloroform.

11. 70% and 100% ethanol.

12. Tabletop microcentrifuge, such as Eppendorf 5415D.

13. Magnetic stir plate.

14. Diethylpyrocarbonate (DEPC): Store at 4°C (see Note 8).

15. DEPC-H₂O: Add 0.1% v/v DEPC to ddH₂O and mix overnight on a magnetic stir plate at room temperature, autoclave at 121°C for 25 min (see Note 9).

16. 10x DIG labeling mix (Millipore Sigma): Store at −20°C (see Note 10).

17. RNase inhibitor (Promega, 40U/μl): Store at −20°C.

18. T3 (Millipore Sigma), T7 (Millipore Sigma) and Sp6 (Millipore Sigma) RNA polymerases: Store at −20°C.

19. DNaseI (RNase free, Millipore Sigma): Store at −20°C.

2.4. Materials for RNA in situ hybridization

The amount of reagents in this section is enough for processing five slides in a 50 ml Coplin jar, and should be adjusted according to the number of slides and size of the container used.

For Day 1: Hybridization of cryosections on the slides

All containers and reagents need to be RNase-free on Day 1.

1. Coplin jars: 50 ml.

2. Diethylpyrocarbonate (DEPC): Store at 4°C (see Note 8).

3. Magnetic stir plate.

4. DEPC-H₂O (400 ml): add 400 μl DEPC to 400 ml ddH₂O. Mix overnight on a magnetic stir plate at room temperature, and autoclave at 121°C for 25 min (see Note 9).

5. DEPC-PBS (400 ml): add 400 μl DEPC to 400 ml PBS. Mix overnight on a magnetic stir plate at room temperature, and autoclave at 121°C for 25 min (see Note 9).

6. 4% PFA in DEPC-PBS (80 ml): Thaw two 10 ml aliquots of 16% PFA (see section 2.1 for 16% PFA preparation) and dilute each with 30 ml DEPC-PBS. Store at 4°C.

7. 0.25% acetic anhydride in 0.1M TEA-HCl (40 ml): add 0.742 g Triethanolamine-HCl and 360 μl 5 N NaOH to 39.8 ml DEPC-H₂O to make TEA-HCl. Add 100 μl acetic anhydride right before use (see Note 11).

8. 20 μg/ml Proteinase K (proK; Millipore Sigma): Dissolve one vial of proK (100mg) in 5 ml DEPC-H₂O to make 20mg/ml stock. Aliquot and store at −20°C. Thaw an aliquot before use and add 40 μl into 40 ml DEPC-PBS (see Note 12).

9. 70% ethanol in DEPC-H₂O (40 ml): 28 ml 100% ethanol, 12 ml DEPC-H₂O.

10. 95% ethanol in DEPC-H₂O (40 ml): 38 ml 100% ethanol, 2 ml DEPC-H₂O.

11. Hybridization solution (40 ml): mix the following in a clean 50 ml tube, aliquot and store at −20°C (see Note 13).

    20 ml	formamide (deionized, aliquot in 50 ml tubes and store at −20°C)
    8 ml	50% dextran sulfate
    400 μl	100x Denhardt’s (VWR, aliquot in 1.5 ml tubes and store at −20°C)
    1 ml	tRNA (10 mg/ml, aliquot in 1.5 ml tubes, store at −20°C)
    2.4 ml	5 M NaCl
    800 μl	1 M Tris-HCl (PH 8.0)
    400 μl	0.5 M EDTA
    400 μl	1 M NaPO4
    4 ml	10% Sarcosyl
    2.6 ml	DEPC-H2O

12. Parafilm (cut into 24 mm × 50 mm pieces) or RNase-free plastic coverslips.

13. A hybridization oven.

14. A humidified slide incubation chamber: see section 2.2.

For Day 2: Post-hybridization washes and antibody incubation

Starting from day 2, reagents do not need to be RNase-free.

1. 20x saline-sodium citrate (SSC) buffer: mix the following in an appropriate container

   • 800 ml ddH₂O
   • 175.3 g NaCl
   • 88.2 g Sodium Citrate

   Adjust PH to 7.0 with a few drops of 12 N HCl. Adjust the volume to 1 liter with ddH₂O. Aliquot and autoclave at 121°C for 25 min.

2. 5x SSC (40 ml): 10 ml 20x SSC, 30 ml ddH₂O.

3. 2x SSC (40 ml): 4 ml 20x SSC, 36 ml ddH₂O.

4. 0.1x SSC (40 ml): 0.2 ml 20x SSC, 39.8 ml ddH₂O.

5. High Stringency Wash Buffer (120 ml) (Make fresh): 60 ml formamide, 12 ml 20x SSC, 48 ml ddH₂O.

6. PBT (350 ml, enough for both day 2 and 3): add 350 μl Tween 20 to 350 ml PBS in a 500 ml bottle. Mix well by shaking vigorously. Alternatively, 10% Tween 20 stock can be made in ddH₂O in advance and stored at room temperature.

7. RNase buffer (400 ml)(see Note 14): mix the following

   40 ml	5 M NaCl,
   4 ml	1 M Tris-HCl, pH 7.5,
   4 ml	0.5 M EDTA, pH 8.0,
   352 ml	ddH2O

8. 10 mg/ml RNase A stock (DNase free): add 10 ml 0.01 M NaOAc (pH 5.2) to a vial of 100 mg RNase A. Heat at 100°C for 15 min. Cool to room temperature. Add 1 ml 1 M Tris•HCl (pH 7.5) and aliquot. Store at −20°C (see Note 15). On the day of experiment, thaw one aliquot and make 20 μg/ml RNase A in RNase buffer by adding 80 μl 10 mg/ml RNase A stock to 40 ml RNase buffer.

9. Alkaline Phosphatase-conjugated anti-Digoxigenin antibody (Fab fragments from sheep, Millipore Sigma): Store at 4°C.

10. A humidified slide incubation chamber: see section 2.2

For Day 3: Color reaction

1. NTMT (80 ml) (Make fresh): see Note 16.

   8 ml	1 M Tris 9.5
   4 ml	1 M MgCl2
   1.6 ml	5 M NaCl
   800 μl	10% Tween20
   40 mg	Levamisol
   Adjust to 80 ml with ddH2O

2. Levamisol: make 50 mg/ml stock in ddH₂O, aliquot and store in 1.5 ml tubes at −20°C.

3. BM Purple (Millipore Sigma): Store at 4°C.

4. Water-based mounting medium, such as Mount Quick (VWR). Store at 4°C

5. Coverslips, such as VWR, 24×50 mm.

6. Nuclear fast red solution (100 ml):

   Dissolve 5 g aluminum sulfate in 100 ml ddH₂O, then add 0.1g nuclear fast red. Boil and stir on a heated magnetic stir plate to dissolve Nuclear fast red. Filter the solution right before use.

3. Methods

Conduct all procedures at room temperature unless otherwise specified.

3.1. Mouse brain cryosection preparation:

1. Dissect embryos in ice-cold PBS in a 6 cm dish (see Note 17).

2. Cut the embryos at the shoulder level and transfer the heads into a 24-well plate with a spoon.

3. Fix the embryos in 4% PFA on a nutator at 4°C for 1 hr for IHC or overnight for RISH.

4. Rinse the embryos with PBS, and then wash the embryos in PBS on a nutator overnight at 4°C.

5. Immerse the embryos in 30% sucrose overnight at 4°C, on a nutator (see Note 18).

6. Change the 30% sucrose and incubate for another 2–3 hrs for further infiltration at 4°C, on a nutator.

7. Transfer the brain samples into a disposable embedding mold and immerse the embryos in O.C.T. compound for 1 hr at 4°C (see Note 19).

8. Position the samples at desired orientation and freeze them on dry ice, or alternatively, in a paperboard box floating on the surface of liquid nitrogen. Wait for 5 min (see Note 20).

9. Transfer the frozen O.C.T. block containing the brain samples to the cryostat and wait at least 1 hr so that the temperature of the block can reach the optimal cutting temperature (see Note 21).

10. Cut 10 μm sections using a cryostat (see Note 22). Collect sections on Superfrost Plus slides.

11. Dry the slides in a micro slide holder for 1 hr at room temperature. Store them in a slide box at −80°C (see Note 23).

3.2. Immunostaining on mouse brain sections

1. Remove sections from −80°C freezer and dry slides in a micro slide holder at room temperature for about 45 min (see Note 24).

2. Place the slides in a Coplin jar and incubate the sections with PBS plus 0.1% Triton X-100 for 1 hr.

3. Take slides out, wipe the backside (the one without sections) and edges with paper towel. Place them with the front side (the one with sections) up on the pipettes of the humidified slide incubation chamber (Figure 1; see Note 25).

4. Apply primary antibodies to the sections (diluted in blocking buffer, 300 μl per slide), and incubate overnight at 4°C (see Note 26).

5. Pour primary antibody onto a paper towel and place the slides in a Coplin jar. Wash the slides with PBS plus 0.1% Triton X-100, 3 × 10 min.

6. Similar to step 3, place slides in the humidified chamber and incubate in the dark with appropriate fluorophore-conjugated secondary antibody (diluted in blocking buffer, 300 μl per slide) for 2 hr (see Note 27).

7. Pour secondary antibody onto a paper towel and place the slides in a Coplin jar. Wash with PBS plus 0.1% Triton X-100, 3 × 10 min.

8. Take slides out and wipe the backside and edges with paper towel. Place them into a micro slide tray.

9. Apply 30 μl Dabco evenly onto each slide and mount with a coverslip (see Note 28 and 29); seal the slides with nail polish.

10. Observe the sections under a fluorescence microscope and take photos with a cooled CCD camera. Some examples are shown in Figure 2

11. Slides can be stored at 4°C protected from light (see Note 30).

Figure 2.

Immunostaining of brain sections. Shown are two coronal sections of an E12.5 brain incubated with a monoclonal antibody against Pax6 and a Cy3-conjugated secondary antibody. (A) Pax6 is weakly expressed in the dorsal region and strongly expressed in the intermediate region in the hindbrain. (B) Pax6 is expressed in the diencephalon (Di), but not the Mesencephalon (Mes). Dashed line demarcates the boundary between the Diencephalon and Mesencephalon.

3.3. Synthesis of RNA probes for RISH

1. Clone the cDNA (or part of the cDNA if the full-length cDNA is longer than 1500 bps; see Note 31) of the gene of interest into a plasmid containing the promoters for the viral RNA polymerases (T3, T7 and Sp6; such as pBluescript).

2. Linearize the template:

   Choose a unique restriction site in the multiple cloning sites on the 5’ end of the cDNA. Cut ~10 μg DNA in 20–50 μl reaction with the corresponding restriction endonuclease overnight using at least 20 IU enzyme. Run small amount (0.1–0.5 μg) in a 0.8% agarose gel to check the efficiency of restriction enzyme digestion.

3. Purify the linearized DNA with a DNA clean up or PCR purification kit (such as The Nucleospin Gel and PCR clean up kit from Macherey-Nagel) and elute in 30 μl elution buffer (final concentration is ~0.3 μg/μl). The linearized template can be stored at −20°C

4. In vitro transcription:

   2 μl	10x transcription buffer
   2 μl	DIG labeling mix
   6 μl	linearized template DNA
   0.5 μl	RNase inhibitor
   2 μl	RNA polymerase (T3, T7 or Sp6; use the one whose promoter is at the 3’ end of the cDNA)
   7.5 μl	DEPC-H2O
   Incubate at 37°C for 2 hrs.

5. Run 1μl in a 1% agarose gel for 20 min to 1 hr to check the yield of the probe.

6. DNase I treatment (optional):

   Add 1μl DNase I and 1μl RNase inhibitor to the RNA probe, incubate at 37°C for 15 min to remove the template.

7. Add 180 μl DEPC-H₂O, 20 μl 3 M NaOAc, mix well. Then add 600 μl 100% ethanol, mix well, and leave at −80°C for 30 min.

8. Centrifuge at top speed (>14,000g) in a tabletop centrifuge for 15 min at 4°C (see Note 32).

9. Discard the supernatant and rinse the pellet with 70% ethanol once without disturbing the pellet.

10. Discard 70% ethanol and dry the pellet for 5 min. Dissolve the RNA probe in 40–50 μl DEPC-H₂O and store at –80°C.

3.4. RNA in situ hybridization of mouse brain sections

Day 1: Hybridization of cryosections (all steps are carried out in Coplin jars unless otherwise specified; avoid RNase contamination)

1. Dry slides at room temperature for about 45 min (see Note 24).

2. Post-fix slides in 4% PFA in DEPC-PBS for 10 min (see Note 33).

3. Wash with DEPC-PBS, 2 × 5min.

4. Drain excess DEPC-PBS and incubate for 6 min in 20 μg/ml proteinase K in DEPC-PBS (see Note 34).

5. Drain and wash with DEPC-PBS for 5 min.

6. Re-fix in 4 % PFA for 5 min, then wash 5 min in DEPC-PBS (see Note 35).

7. Acetylate sections with acetic anhydride in 0.1 M TEA-HCl for 10 min (see Note 36).

8. Wash in DEPC-PBS for 5 min, dehydrate in 70% ethanol for 5 min and 95% ethanol for 2 min. Air dry for 30 min to 2 hr.

9. Add 2 μl RNA probe (approx. 1 μg) to 1 ml hybridization solution and heat at 80°C for 2 min (see Note 37).

10. Place slides horizontally in a humidified slide incubation chamber. Cover sections with 200 μl of hybridization solution with the probe and lower parafilm coverslips over sections avoiding bubbles (see Note 38).

11. Seal the slide incubation chamber carefully and hybridize at 55°C in a hybridization oven overnight (16–18 hrs).

Day 2. Post-hybridization washes and antibody incubation (all steps are carried out in Coplin jars; RNase-free environment is not required)

1. Dip slides gently in a Coplin jar filled with 5x SSC to let the coverslips float off the slides (see Note 39).

2. Incubate the sections in high stringency wash at 65°C in a Coplin jar for 30 min (see Note 40).

3. Wash in RNase Buffer at 37°C, 3 × 10 min.

4. Wash in RNase Buffer with 20 μg/ml RNase A at 37°C for 30 min (see Note 41).

5. Wash in RNase buffer at 37°C for 15 min.

6. Repeat high stringency wash (as in step 2) at 65°C, 2 × 20 min.

7. Wash in 2 × SSC, then 0.1 × SSC for 15 min each at 37°C.

8. Wash with PBT for 15 min.

9. Take the slide out of PBT, wipe the backside and edges of the slide with paper towel. Place slides horizontally in a humidified slide incubation chamber and block for 1 hr with 10% goat serum in PBT (300 μl per slide).

10. Pour the blocking buffer onto paper towels. Wipe the backside and edges of the slide with paper towel. Incubate with AP-conjugated anti-Digoxigenin antibody (diluted 1/5000 in PBT with 1% goat serum, 300 μl per slide) at 4°C overnight in the same humidified chamber.

Day 3: Color reaction

1. Pour the antibody onto paper towels. Place slides in a Coplin jar and wash in PBT for 5 times, 1 hr each.

2. Wash 2 × 10 min in freshly prepared NTMT buffer.

3. Wipe the backside and edges of the slide with paper towel. Place slides horizontally in a humidified slide incubation chamber and incubate overnight to several days in BM purple solution (300 μl per slide) supplemented with 0.5 mg/ml Levamisol in the dark (see Note 42).

4. Observe periodically the progress of the color reaction under a microscope. If the staining is not ready, re-apply BM purple solution and incubate for longer time (see Note 43).

5. When the signal is strong and the background staining just begins to show, place slides back into a Coplin jar. Wash slides in PBS for 2–5 min and dip briefly in ddH₂O (see Note 18).

6. (optional) Counterstain the sections with Nuclear fast red until the sections turn slightly pink. Usually it takes 2–3 min.

7. Wash excess Nuclear fast red in slow-running tap water.

8. Wipe the backside and edges of the slides with paper towel. Apply mounting medium to the slides and put coverslips on (see Note 44).

9. Observe the staining under a microscope and take photos with a color camera. Some examples are shown in Figure 3.

10. The mounted sections can be stored at 4°C.

Figure 3.

RNA in situ hybridization of brain sections. Shown are two sections of E12.5 brains hybridized with a Digoxigenin-labeled RNA probe against Otx2. (A) A coronal section through the forebrain region shows Otx2 expression in ventral telencephalon (tel) and the retinal pigment epithelium of the eye, but not in ventral Diencephalon (di). (B) A sagittal section shows that Otx2 is expressed in the Mesencephalon (mes), Diencephalon (di), but not in the Metencephalon (met) and Pons. The arrowhead points to the boundary between Diencephalon and Mesencephalon. Dashed lines demarcate the boundary between the midbrain and hindbrain. is: Isthmus.

4. Notes:

1. The 24-well plates are for the convenient storage of individual small sample, such as the brains of E12.5 embryos or younger. Vials of appropriate size should be used for older/bigger brain samples.

2. Distilled, de-ionized water (ddH₂O) used in this protocol is ultra-purified water with a resistance of 18.2 MΩ.

3. 16% PFA should be prepared in a fume hood because PFA is highly volatile and irritant.

4. It is critical to use Superfrost Plus slides as they contain a special coating to prevent the sections from falling off the slides during the incubation and washing steps.

5. Make fresh blocking buffer for each experiment. Do not store for more than a week.

6. Dabco is irritant. Avoid contact with skin and eyes. Avoid inhalation of vapor or mist.

7. Ethidium is highly carcinogenic. Avoid direct contact and dispose Ethidium-containing waste properly.

8. DEPC is toxic. Avoid direct contact.

9. All DEPC-treated solutions need to be autoclaved to degrade DEPC, which can react with RNA.

10. When thawing the DIG-labeling mix, avoid prolonged incubation at 37°C to reduce the chance of NTP degradation.

11. Keep acetic anhydride from moisture (keep the container tightly closed all the time) and add acetic anhydride immediately before use.

12. Do not re-freeze proK for RNA in situ hybridization. The activity of proK should be tested every time a new batch is introduced.

13. To make 50% dextran sulfate, mix 10 ml dextran sulfate and 10 ml ddH₂O in a 50 ml tube by inverting, shaking, vortexing and heating at 60°C. When dextran sulfate starts to get into water, the total volume will decrease. Add more ddH₂O to keep the final volume at 20 ml.

14. Sterile RNase buffer can be stored at room temperature.

15. Take caution to avoid contaminating the bench and other lab materials with RNase A.

16. NTMT should be freshly prepared.

17. At this step, we usually remove the extra-embryonic membranes or the tips of the tails to genotype the embryos.

18. The tissue should float on the surface of the 30% sucrose initially, and sink to the bottom of the well after sucrose has fully infiltrated the tissue. Make sure the tissue is completely immersed in the sucrose solution, as the morphology of tissues staying at the air/liquid interface can be distorted by surface tension.

19. The time the embryos immersed in OCT depends on the size and density of the tissue. For large and/or dense tissue, longer time is needed.

20. Do not freeze samples by directly placing the embedding mold in liquid nitrogen.

21. If not cutting sections right away, wrap the embedding mold with parafilm and keep it at −80°C for up to one week.

22. See manufacturer’s manual for how to cut cryosections.

23. Cryosections can be stored for months at −80°C without noticeable degradation of proteins and mRNAs.

24. Dried sections do not fall off the slides in subsequent experiments. In addition, drying creates holes in the subcellular structure, permeabilizing the cells for further experiments.

25. Take caution not to let the sections dry completely. If necessary, process slides one at a time. This applies to all steps that involve taking slides out of the solution.

26. The antibody solution stays on the top of slides only if the edges and the bottom of the slides are dry. Therefore, add the antibody solution to the center of the slide and avoid moving the chamber once the antibody is added. Place a sign on the chamber to warn others not to move it during incubation.

27. Covering the humidified slide incubation chamber with foil or perform the incubation in a cabinet.

28. Cut the pipet tip to make a large orifice because Dabco is sticky.

29. To avoid bubbles, lower the coverslip slowly.

30. The fluorescent signal decreases over time, so try to observe the fluorescence as early as you can.

31. In general, shorter probes (<500 bps) tend to yield weaker signals, whereas very long probes (>1500 bps) have difficulty penetrating the cell membranes.

32. Take caution when removing the supernatant because the pellet may not be visible.

33. Post-fixation ensures the tissue is fixed equally with cross-linked RNA molecules. It also improves the retention of the tissue on the slide.

34. To drain excess DEPC-PBS, hold the slides and tilt the Coplin jar onto the paper towels. Proteinase K treatment improves the signal intensity by allowing greater access of the target mRNA for the probes.

35. Re-fixation improves the section stability after proteolytic digestion.

36. Acetylation chemically modifies proteins and reduces their non-specific bindings.

37. Preheat the hybridization solution at 80°C before use.

38. To avoid bubbles, lower the parafilm coverslips slowly from one side to the other. Take caution when transporting the slide chamber to the oven such that the coverslips do not fall off the slides.

39. Don’t force the coverslips off the slides with forceps, or the sections may tear.

40. Pre-warm solution for this step and steps 3–7. During the high-stringency wash, low salt concentration and high temperature inhibits non-specific bindings.

41. RNase A digests single-stranded RNA to reduce the background signal.

42. Wrap the slide chamber in foil or place the chamber in a dark cabinet.

43. Before checking the staining status of the sections, prepare BM purple solution with 0.5 mg/ml levamisol in case the staining is not ready and more incubation with BM purple is needed. Otherwise, the sections may become dry before the BM purple solution is ready.

44. The mounting medium is very sticky and solidifies quickly. It is better to apply the mounting medium before the slides dry and put coverslip on immediately.