Protocol for measuring mitochondrial size in mouse and human liver tissues

Summary

Mitochondrial dynamic process is important for cell viability, metabolic activity, and mitochondria health. Here, we present a protocol for measuring mitochondrial size through immunofluorescence staining, confocal imaging, and analysis in ImageJ. We describe the steps for tissue processing, antigen retrieval, mitochondrial staining using an integrating immunofluorescence assay, and computerized image analysis to measure each mitochondrial size in mouse and human liver tissues. This protocol reduces tissue sample volume and processing time for the preparation of primary cells.

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

Graphical abstract

Highlights

• •Protocol for measuring mitochondrial size in mouse and human liver tissues
• •Detailed step for mitochondrial staining using immunofluorescence assays
• •Use high-pressure cooker for antigen retrieval to achieve high-quality images
• •Quantify individual mitochondrial size using ImageJ software

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

Mitochondrial dynamic process is important for cell viability, metabolic activity, and mitochondria health. Here, we present a protocol for measuring mitochondrial size through immunofluorescence staining, confocal imaging, and analysis in ImageJ. We describe the steps of tissue processing, antigen retrieval, mitochondrial staining using an integrating immunofluorescence assay, and computerized image analysis to measure each mitochondrial size in mouse and human liver tissues. This protocol reduces tissue sample volume and processing time for the preparation of primary cells.

Before you begin

To maintain a healthy mitochondrial population, mitochondria continually assume fusion and fission processes.² Even though, mitochondria fusion will allow complementation of dysfunctional components in different mitochondrion, however, this will lead to decreased bioenergetic capacity and decreased workload of nutrient metabolism.^3,4,5 Mitochondrial fission can also promote the removal of damaged mitochondria by mitophagy,² thus, to maintain a healthy mitochondrial population, which is especially important under excessive nutrient.^6,7

Patients with nonalcoholic fatty liver disease (NAFLD) have giant mitochondria (megamitochondria)^5,8 and megamitochondria are evenly distributed in all zones within the liver of patients with NAFLD.⁹ Mitochondrial size in human’s liver increases with age, especially after 60 years of age.¹⁰ Animal study showed that increased mitochondrial size exhibited impaired mitochondrial metabolism.¹¹

In our recent publication, we validated these reports and found that, in primary hepatocytes of young mice or lean healthy mice, the majority (>80%) of the mitochondria were in a shortened form. However, mitochondria became elongated and formed reticula with aging and under obesity. Importantly, promotion of mitochondria fission in hepatocytes with elongated mitochondria improves mitochondrial oxidative activity, reduces the reactive oxygen species, and eliminates compromised mitochondria. Therefore, mitochondrial size can be used as a biomarker of mitochondrial activity.¹

Here, we present a technique using immunofluorescence staining with specific antibodies against mitochondrial matrix protein pyruvate dehydrogenase (PDH) or mitochondrial outer membrane protein TOM20 to accurately measure mitochondrial size in frozen or paraffin-embedded liver tissues. Using this technique, we validated that the average mitochondrial size increased more than 3-fold in the liver of elderly mice compared to the liver of young mice, and significantly increased in the liver of obese patients compared to lean healthy individuals.¹ This technique overcomes the difficulty in acquiring large amounts of tissues and time-consuming procedures for the preparation of primary cells.

Institutional permissions

Animal studies presented were approved by the Johns Hopkins University Animal Care Ethics Committee. The use of human samples was approved by Institutional Review Board of Johns Hopkins School of Medicine.

Reagent preparation for collecting liver tissue

Timing: 1–2 days

Note: The present protocol is determined on assessing the mitochondrial size from mouse and human liver. After isolation of the liver tissue from mice, it is important to process liver tissue immediately or liver tissue needs to be snap-frozen in liquid nitrogen.

• 1.
  Prepare 4% (w/v) Paraformaldehyde (PFA).

  CRITICAL: Avoid exposure to eye and skin, as per institutional guidelines handling. Paraformaldehyde powder should always be used in a ventilated safety hood.

  • a.
    Weigh 20 g of Paraformaldehyde and add it to a flask with a magnetic stirrer rod and 450 mL of 1× PBS.
  • b.
    Stir constantly and heat the mixture to a temperature of 60°C.
  • c.
    The PFA solution is heated to 60°C and bring it to a clear solution by adding 100 μL of 5 N NaOH.
  • d.
    Turn off the heat and adjust the pH to 7.4, to make a volume of 500 mL with 1× PBS.
  • e.
    Filter the solution by Whatman filter paper, aliquot it and freeze for long-term storage.
• 2.
  Prepare 15% (w/v) sucrose and 30% (w/v) sucrose.

  • a.
    Weigh 30 g of sucrose and make up to 200 mL with 1× PBS.
  • b.
    Weigh 60 g of sucrose and make up to 200 mL with 1× PBS.
• 3.
  Prepare Ethanol with different percentage (100%, 95%, 90%, 80% 70% (v/v))

  • a.
    For 95%, measure the 95 mL of ethanol, and make up to 100 mL with Milli-Q water.
  • b.
    For 80%, measure the 80 mL of ethanol, and make up to 100 mL with Milli-Q water.
  • c.
    For 70%, measure the 70 mL of ethanol, and make up to 100 mL with Milli-Q water.
• 4.Prepare 1 mM EDTA.

Add 0.2 mL of 0.5 M EDTA to 99.8 mL of Milli-Q water to achieve the 1 mM EDTA solution.

• 5.High Pressure cooker.

Fill the cooker with 1000 mL water up to the line marked in the cooker.

• 6.Prepare PBS-T (0.05% (v/v).

Add 250 μL of Tween-20 and make up to 500 mL with 1× PBS.

• 7.Prepare 3% (w/v) BSA.

Weigh 3 g of bovine serum albumin powder, and make up to 100 mL with 0.05% (v/v) PBS-T.

Collecting liver tissue

Timing: 1 day

Note: Liver tissue can be collected with or without fixation with 4% (w/v) Paraformaldehyde.

• 8.Collection of liver tissue.

Dissect the liver and cut the liver tissue into the size 0.3 cm × 0.3 cm and use immediately in the following step. Otherwise, snap frozen the cut pieces in liquid nitrogen for long-term storage.

• 9.
  Collection of liver tissue after fixation with 4% (w/v) Paraformaldehyde.

  • a.
    Anesthetize the mice by intraperitoneal injection of ketamine/xylazine (75 and 10 mg/kg, respectively).

    Note: We used ketamine and xylazine to anesthetize the mice; the combination of these two drugs provides a faster and smoother induction, good muscle relaxation. However, this protocol should work with tissue collected in mouse that was sacrificed in a different method.

  • b.
    Mice will be fixed by perfusing ice-cold 4% (w/v) Paraformaldehyde via cardiac puncture, and then, liver will be dissected.

    Note: Liver perfusion with PFA will allow the delivery of fixative reagent directly to tissue capillary beds to fix tissues before autolysis begins and make them less susceptible to artifacts. Based on our experience, this method provides the best mitochondrial morphology. Furthermore, for achieving high quality mitochondrial morphology, PFA works well when compared to other fixative solutions.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Anti-PDHA1 mouse monoclonal	Abcam	Cat# ab110330
TOM20 polyclonal	Proteintech	Cat# 11802-1-AP
Goat anti-mouse IgG (H + L)	Thermo Fisher Scientific	Cat# A-11004
Goat anti-rabbit IgG (H + L)	Thermo Fisher Scientific	Cat# A-11034
DAPI	Thermo Fisher Scientific	Cat# 62248
Chemicals, peptides, and recombinant proteins
BSA	Sigma	Cat# A4503-100G
Sucrose	Sigma	Cat# S0389-500G
Paraformaldehyde	Sigma	Cat# P6148-500G
Tween 20	Sigma	Cat# P9416-100ML
1× PBS	Quality Biological	Cat# 114-058-101CS
Ethanol	Fisher Scientific	Cat# FSA4094
Xylene	Sigma	Cat# 534056-4L
EDTA	Sigma	Cat# E9884-500G
Experimental models: Organisms/strains
Mouse: C57BL/6	In-house	8–10 weeks old male mice
Software and algorithms
NIS-Elements
ImageJ	NIH	https://imagej.nih.gov/ij/
GraphPad Prism
Other
Pap pen	Abcam	Cat# ab2601
Mounting media	Sigma	Cat# 06522-100 ML
Coverslip (24 × 50 mm)	Fisher Scientific	Cat# 12544E
Whatman filter paper	GE Healthcare Life Sciences	Cat# 1001-240
Plastic case	Fisher Scientific	Cat# NC0731404

Materials and equipment

Reagent or resource	Final concentration	Amount
4% Paraformaldehyde
Paraformaldehyde Powder	4%	20 g
5 N NaOH	0.02%	100 μL
Milli-Q water	N/A	450 mL
Immunofluorescence Blocking solution
BSA	3% (m/v)	3 g
Tween-20	0.05% (v/v)	100 μL
Make fresh and store at 4°C for up to a week
Primary Antibody Staining solution (Primary antibodies were dissolved in PBS-T (0.05%))
Anti-PDHA1 mouse monoclonal	1:400	1.25 μL
Tom20 Polyclonal	1:200	2.5 μL
DAPI	1:1000	0.5 μL
Make immediately prior to staining
Secondary Antibody Staining solution (Secondary antibodies were dissolved in 1× PBS)
Goat anti-Mouse IgG (H + L)	1:200	2.5 μL
Goat anti-Rabbit IgG (H + L)	1:200	2.5 μL
Make immediately prior to staining

Step-by-step method details

Tissue section fixation with Paraformaldehyde

Timing: ∼2 days

As we mentioned above, this protocol will outline the methods to visualize individual mitochondria size in the liver tissue. Before going to section the tissues, the following steps are needed for getting a clear morphological structure.

• 1.After removal of liver tissue from mice, fix the tissues in fresh 4% Paraformaldehyde at 22°C for 24 h.

Note: To get the best morphology of the tissues, it must be fixed for a minimum of 24 h and maximum 48 h. Fixation time will differ based on the specimen size.

• 2.Remove the tissue from Paraformaldehyde solution and place it in 15% sucrose in PBS until the tissue sinks.

Note: For best results, tissue must be immersed in the sucrose for minimum 6–12 h.

• 3.Remove the tissue from 15% sucrose, place it in 30% sucrose in PBS until the tissue sinks and keep it 16 h at 22°C.
• 4.Replace the 30% sucrose with 70% ethanol.

Note: The tissue which is immersed in 70% ethanol is sent for paraffin embedded tissue processing and sectioning to Johns Hopkins histology core facility and instructed to process the tissue section at 7 μm thickness.

De-paraffinize (remove paraffin from the tissue sections)

Timing: ∼45 min

• 5.Place the slides in plastic slide case and fill it with xylene until the tissue section is immersed and leave for 5 min. Repeat this step 2 times.

Note: This step needs to be performed in ventilation hood.

• 6.After xylene incubation, put the slides in 100% ethanol and incubate at 22°C for 3 min. Repeat this step again.

CRITICAL: The aim of this step is to remove the xylene from the slides. Because xylene dissolves in ethanol and not in water, xylene must be completely removed before further processing.

• 7.Wash the slides with Milli-Q water for 5 min.
• 8.Place the slides in the order of 95%, 80% and 70% Ethanol each 1 time for 1 min.
• 9.Wash the slides with Milli-Q water for 5 min. Repeat this step 2 times.

Note: Change the Milli-Q water for each wash.

Antigen retrieval by using high pressure cooker

Timing: ∼30 min

• 10.
  Place the slides in a plastic case containing 1 mM EDTA.

  • a.
    Keep the slides in the high-pressure cooker.
  • b.
    Set the pressure on high for 15 min.
  • c.
    Then, start the antigen retrieval.

CRITICAL: We used the Cuisinart pressure cooker (Cat#CPC-600) (Figure 1). After 15 min of high pressure, don’t open the steamer until the pressure releases completely. Once the complete pressure is released from the cooker, take the slides out and keep the slides at 22°C, allowing the slides to cool for 15 min.

Figure 1.

Cuisinart pressure cooker settings screenshot view of the high steamer for antigen retrieval

“Start” Button allows for antigen retrieval processing. While using this feature, users can set the time, high and low pressure.

Blocking of the tissue sections

Timing: ∼60 min

• 11.Blocking with 3% BSA in PBST for 1 h at 22°C.

Note: Before blocking, slides are circled by pap-pen which forms a boundary for the tissues. Add the blocking buffer inside the boundary by which it covers the entire tissue. In addition, we tested different blocking agents, such as, horse serum and Superblock (Thermos Scientific) and found that BSA works well because it contains only one protein, leading to less cross-activity.

Antibody incubation and mounting

Timing: ∼2 days

• 12.Add the 500 μL of primary antibody (1.25 μL of PDH1 or 2.5 μL of TOM20) and 0.5 μL of DAPI together over the specimen marked by the pap pen. Incubate 16 h at 4°C.

Note: Prepare primary antibody (PDH1 and TOM20) with DAPI together and dissolved in 500 μL of PBS. For one slide, 500 μL is sufficient to get an efficient picture. However, optimization is needed based on the specimen size.

• 13.Wash the slides with PBST for 5 min. Repeat this step 2 times.
• 14.Add secondary antibodies dissolved in PBST for 1 h at 22°C.

Note: Prepare secondary antibodies (2.5 μL Goat anti-Mouse IgG (H + L) and 2.5 μL Goat anti-Rabbit IgG (H + L)) in 500 μL of PBST.

• 15.Wash the slides with PBS for 5 min. Repeat this step 2 times.
• 16.Mount the slides by adding antifade mounting medium and cover with a coverslip.

CRITICAL: Use 30 μL of mounting medium on specimen. Place the edge of the cover slip over the sample and carefully lower the coverslip into place using a 1 mL pipette tip or equivalent. This will help to prevent the formation of air bubbles under the coverslip.

Image acquisition of individual mitochondria structure by confocal microscopy

Timing: ∼1 h

• 17.Turn on the confocal microscopy and open the Nikon NIS elements software.
• 18.Place the slide onto a 20× lens to view and locate the mitochondria.

Note: “Scan” button allows to view the image. Confirm the staining of the cells by DAPI. Adjust the laser intensity, emission, gain, offset and pinhole size, using NIS elements C2plus GUI image settings to get the efficient quality. Follow Figure 2 for image settings and optimization.

• 19.View the image to check the noise and background. If the settings are good, then change the lens from 20× to 60× with a drop of immersion oil.
• 20.Check once again all image parameter settings are ideal before starting the scan to capture the mitochondria.
• 21.To generate the quality mitochondria images, use the Z-stack with three channels (DAPI, PDH1, TOM20) for the images with slice intervals of 0.16 μm, maximum of 10–12 slices.

Note: To focus the z-plane to start and end the z-plane, open the software and select the “Z-stack” tab and follow the start button to begin the z-stack. Using the “focus” button to adjust the focus of the microscope to the desired starting point of the z-stack. Then, click the “add” button to add the current z-plane to the stack and use the focus button to adjust the focus of the microscope to the next z-plane. Repeat the steps, until you have added all the z-planes intended to include in the stack.

• 22.All images should be saved in .nd2 format and perform the analysis process.

Note: During the analysis process, make sure all images are in the same settings including scale size and save in the .tiff format.

Note: In the menu C2plus compact GUI option, mitochondria were imaged using corresponding filters to each secondary fluorescence antibodies and DAPI (Figure 2). Mitochondria require high signal-to-noise ratio images. To achieve this, 60× objective lens can be used and matched with the large-format 1024 pixel with 1/8 fast mode.

Figure 2.

Nikon confocal imaging of mitochondria settings-screenshot view of the NIS elements software control panel with parameters used to image the mitochondria

“Scan” button allows for live viewing of staining in real time. While using this feature, users can set the DAPI, FITC and Alexa546. “Channels” parameters are used to modify pinhole, channel gain, laser selection, and other imaging parameters for each individual channel.

Image analysis

Timing: ∼30 min

• 23.Open the image in ImageJ software select the following: image>color>split channels.
• 24.Then select the image>adjust>threshold to visualize the mitochondrial size and staining. Note: Adjust the threshold for all images including three channels in uniform manner.
• 25.Before analyzing mitochondrial size, follow Figure 3 for set scale and set measurements, and analyze particles in the size of μm.

Note: The threshold column: we used the default threshold algorithm and adjust the upper lane value in the rage 162 and lower value is 255 with the similar select option of dark background and don’t reset range. Process and analyze the images in ImageJ software and set the default scale with μm and make pixel distance 1.6, known distance and pixel ratio is 1.

Figure 3.

The parameter settings of ImageJ software to analyze the mitochondrial size

Expected outcomes

Using this method, we determined mitochondrial size in the liver of young and elderly mice and found that the average mitochondrial size increased 3 times in elderly mice compared to mitochondrial size in the young mice (Figure 4) (Table S1). These data are in agreement with previous report that mitochondrial size in the liver increases with age.¹⁰ Obese patients have giant mitochondria in the liver.^5,8,9 We also observed that obese patients have significantly larger mitochondrial size in the liver compared to healthy individual (Figure 5) (Table S2). For images of liver mitochondrial staining with TOM20 antibody, please refer to our recent publication.¹

Figure 4.

Cryosections of livers from 8-week young and 78-week elderly mice were subjected to the immunofluorescence staining with PDH antibody (left and middle panels) and mitochondrial size was determined (right panel)

Scale bar, 10 μm.

Figure 5.

Cryosections of livers from healthy individuals’ sample and obese patients’ sample were immunofluorescence stained with PDH antibody (left and middle panels) and the mitochondrial size was determined (right panel)

Scale bar, 10 μm.

Limitations

The protocol was designed and optimized to quantify the individual mitochondrial size in the liver tissue. This protocol serves as a suitable starting point for fixation, tissue processing, staining and image acquisition and analysis of mitochondria. However, the protocol as presented requires validation in other tissues based on their morphological, thickness and experimental applications. To achieve a better mitochondria morphology, this depends on in situ fixation. However, the optimizations of tissue processing, antigen retrieval time and the concentrations of fixative optimization are needed for other tissues to prevent mitochondrial structure damage.

Troubleshooting

Problem 1

The structure of the mitochondria shows poor morphology, shrink and artificial space (Part 1, step 1).

Potential solution

• •Do the fixation and tissue processing immediately after the removal of tissue from mice. It will help to inhibit the metabolic process by enzymes which are present in the tissue. Use sterile consumables and solutions to prevent the contamination and improve the cell structure and tissue autolyzing.
• •Penetration of fixative in the liver tissue takes up to 24 h. Make sure this step is done in a timely manner, and it will differ due to the specimen size. If tissue specimen is too large, optimization is needed for the fixative volume and incubation time.
• •We kept the tissue on 70% ethanol after the fixative and sucrose incubation before processing dehydration, wax infiltration and embedding procedure and sectioning.

Problem 2

Lack of uniform mitochondria staining (Part 3, step 1).

Potential solution

• •Use the Cuisinart pressure cooker in the high-pressure mode with minimum processing time of 15 min until the full pressure release. It will take a minimum of 25 min. The best indication of successful antigen retrieval is acquired when the specimen looks transparent on the slides.
• •To prevent slides and tissue damage always fill the water up to the specific line which is marked in the pressure cooker during the antigen retrieval processing.
• •Take the slides out from the pressure cooker and allow the slides to cool at 22°C for 15–20 min. By avoiding this step, sudden changes in the temperature will lead to tissue damage.

Problem 3

High background staining and poor-quality images (Part 6, step 2).

Potential solution

• •Adding more wash steps after the incubation of primary and secondary antibodies.
• •We optimized the protocol for mouse liver tissue. However, optimizing the primary and secondary antibodies dilution based on specific tissues used for processing is recommended.
• •During the mounting process, avoid air bubbles and excess medium on the specimen slide by gentle handling.

Problem 4

Images poor resolution (Part 6, step 2).

Potential solution

• •Use the laser intensity below 2 in NIS elements software. Adjust each channel with prominent laser intensity, gain, and offset. Scan the images on each individual channel and confirm the quality of resolution. Use the gain below 50 and resolution size 1024 allowing for more sharp and good quality images.

Problem 5

Define the scale bar in ImageJ (part 7, step 3).

Potential solution

• •In confocal microscopy, save the images with 10 μm scale bar. Open the image with scale bar in ImageJ software and select straight line tool and draw an exact length of line over the scale bar. Then choose Analyze' menu select 'Set Scale'. The values like distance in pixels-the known distance will be filled automatically and adjust the units of length. By selecting global option all the images opened later will have the same calibration.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Dr. Ling He, heling@jhmi.edu.

Technical contact

Technical questions on executing this protocol should be directed to and will be answered by the technical contact, Dr. Hiromi Sesaki, hsesaki@jhmi.edu.

Materials availability

This study did not generate new unique reagents.

Data and code availability

The published article includes all details, needed resources, and datasets generated or analyzed during this study.