Materials and reagents

Biological materials

1. Male Wistar rats (2–5 months old, 200–450 g)

Reagents

1. Diethyl ether (Synthesia, catalog number: 60-29-7)

2. Sodium chloride (NaCl) (Sigma, catalog number: S-9625)

3. Heparin, 180 USP units/mg (Merck, catalog number: H3393-10KU)

4. Paraformaldehyde (PFA) (Sigma-Aldrich, catalog number: P6148)

5. Sodium hydroxide (NaOH) (MilliporeSigma, catalog number: S8045)

6. Sodium phosphate monobasic monohydrate (H₂NaO₄P·H₂O) (Sigma-Aldrich, catalog number: 71504)

7. Sodium phosphate dibasic (HNa₂O₄P) (Sigma-Aldrich, catalog number: 04276)

8. Bovine serum albumin (BSA) (Sigma, catalog number: A-7906)

9. Triton X-100 (Sigma, catalog number: T9284)

10. Primary antibodies (Table 1)

11. Secondary antibodies (Table 2)

Table 1. List of tested primary antibodies

Table 2. List of tested secondary antibodies

Solutions

1. 0.9% NaCl (see Recipes)

2. 12% PFA (see Recipes)

3. 0.2 M phosphate buffer (PB) (see Recipes)

4. 10% Triton X-100 (see Recipes)

5. 10% bovine serum albumin (see Recipes)

6. Blocking solution (see Recipes)

7. Antibody dilution solution (see Recipes)

Recipes

1. 0.9% NaCl (w/v)

Note: Prepare the solution immediately before transcardial perfusion to ensure it remains fresh. If needed, store at 4 °C for up to 1 month.

2. 12% PFA (w/v), pH 7.4

Notes:

1. For transcardial perfusion and fixation of tissues, 4% PFA must be used within 48 h after preparation. Therefore, it is better to prepare a 3× stock solution (12% PFA), which can be diluted just before use.

2. 2 M NaOH is used to adjust the pH of solution to 7.4. The volume indicated is approximate.

a. Make all procedures in a fume hood.

b. Dissolve 60 g of PFA in 500 mL of ddH₂O using a magnetic stirrer with a hotplate at 40–50 °C.

Note: The boiling point is 60 °C; be careful not to overheat or boil your solution. To prevent the vaporization of the solution, it is better to cover the glass beaker with foil!

c. For better dissolution, add 2 mL of 2 M NaOH. The solution with fully dissolved PFA is opalescent and nearly transparent.

d. Filter the mixture using a vacuum pump.

e. Store the solution in glassware at 4 °C for up to 3 months.

f. Right before use, dilute the solution three times; the final concentration should be 4%.

3. 0.2 M phosphate buffer (PB), pH 7.4

a. Take 6.9 g of HNa₂O₄P and dissolve it in 250 mL of ddH₂O using a magnetic stirrer at room temperature (RT) to prepare dibasic sodium phosphate.

b. To prepare monobasic sodium phosphate, put 22.72 g of H₂NaO₄P·H₂O inside another glass beaker and dissolve it in 800 mL of ddH₂O using a magnetic stirrer at RT.

c. Adjust the pH of dibasic sodium phosphate to 7.4 with monobasic sodium phosphate by checking it with a pH meter.

d. Store solution at 4 °C for up to 3 months. The formation of crystals at the bottom of your solution after preparation and storage at 4 °C for at least 2 days is normal and indicates that the solution was made correctly.

e. Before usage, heat 0.2 M PB at 37 °C and dilute it to 0.1 M PB with ddH₂O.

4. 10% Triton X-100 (v/v)

Note: Triton X-100 is extremely viscous and difficult to pipette accurately. Preparing the solution may be time-consuming.

a. Add 10 mL of Triton X-100 to 80 mL of ddH₂O in a glass beaker.

b. Use a magnetic stirrer on a low speed setting at RT. Avoid vortexing or high-speed stirring as this will create excessive foam.

c. Once fully dissolved, transfer the solution to a 100 mL graduated cylinder and add ddH₂O to bring the final volume to 100 mL.

d. Store solution at 4 °C for up to 3 months or at -20 °C up to 6 months.

5. 10% Bovine serum albumin (w/v)

a. Add 80 mL of 0.1 M PB to a glass beaker with a magnetic stir bar. Use a magnetic stirrer on a low speed setting at RT.

b. Slowly add 10 g of BSA onto the surface of the stirring PB.

Note: Do not put all the BSA at once, as it will form large clumps that are very difficult to dissolve.

c. Once fully dissolved, transfer dissolved solution to a 100 mL graduated cylinder and add ddH₂O to bring the final volume to 100 mL.

d. Store solution at 4 °C for up to 1 month or at -20 °C up to 6 months.

6. Blocking solution

Notes:

1. All calculations are made per one well of a 24-cell well plate.

2. The volume of the blocking solution should be at least two times the volume of solutions with diluted primary and secondary antibodies for proper blocking, not only to prevent unspecific binding sites on samples but also to block cells on the plate to prevent antibody binding with them.

3. Prepare immediately before usage.

7. Antibody dilution solution

Notes:

1. All calculations are made per one well of a 24-cell well plate.

2. Prepare immediately before usage.

Laboratory supplies

1. 6-well cell culture plates (Falcon, catalog number: 353046)

2. 24-well cell culture plates (Cellstar, catalog number: 662160 or Falcon, catalog number: 353047)

3. Microcentrifuge tubes, 0.6 mL (Fisher Scientific, catalog number: 02-681-311)

4. Microcentrifuge tubes, 1.5 mL (Deltalab, catalog number: 200400P)

5. Pipette tips, 0.1–10 μL (VWR, catalog number: 89041-382)

6. Pipette tips, 2–200 μL (Deltalab, catalog number: 200016A)

7. Pipette tips, 100–1,000 μL (Deltalab, catalog number: 200012)

8. Cell culture dishes, 35 × 10 mm (Cellstar, catalog number: 627160)

9. Filter paper (e.g., Munktell Filtrak, catalog number: 110069)

10. Slice hold-down (Warner Instruments, catalog number: 64-0255)

11. Parafilm M (Parafilm, catalog number: PM-996)

12. Graduated cylinder, 250 mL (any available)

13. Glass beakers, 250 mL, 500 mL, and 1 L (any available)

14. Dissection pad (you can use a foam pad wrapped in aluminum foil)

15. Desiccator (any one available that fits an animal)

16. Cotton wool (any available)

17. Nitrile gloves (any available)

18. Needles (any available)

Equipment

A. Laboratory equipment

1. Pipette, 0.1–2.5 μL (Eppendorf, catalog number: 3123000012)

2. Pipette, 0.5–10 μL (Eppendorf, catalog number: 3123000020)

3. Pipette, 10–100 μL (Eppendorf, catalog number: 3123000047)

4. Pipette, 100–1,000 μL (Eppendorf, catalog number: 3123000063)

5. Scales (any available)

6 Analytical balance (Ohaus, model: VP214C)

7. Magnetic stirrer with hotplate (Joanlab, model: HS5Pro)

8. pH meter (Mettler Toledo, model: FiveEasy Plus)

9. Vacuum pump (Joanlab, model: VP-10L)

10. Laboratory water bath (any available)

11. Fume hood (any available)

12. Peristaltic pump (e.g., Thermo Fisher Scientific, catalog number: 13-876-4)

13. 3D oscillating laboratory shaker (Joanlab, model: OS-30Pro)

B. Tools for transcardial perfusion and isolation of the sciatic nerve

1. Curved iris scissors, 11.5 cm (e.g., World Precision Instruments, catalog number: 501759)

2. Straight operating scissors, 14 cm (e.g., World Precision Instruments, catalog number: 14192)

3. Curved Rochester-Ochsner hemostatic forceps, 16.0 cm (e.g., World Precision Instruments, catalog number: 501710)

4. Scalpel handle (e.g., World Precision Instruments, catalog number: 500237)

5. Scalpel blade, #24 (e.g., World Precision Instruments, catalog number: 500247)

6. Tweezer forceps, 12 cm (e.g., World Precision Instruments, catalog number: 500338)

7. Curved micro scissors, 12 cm (e.g., World Precision Instruments, catalog number: 503364)

C. Imaging

1. Stereomicroscope (Olympus, model: SZX7)

2. Laser scanning confocal microscope (Olympus, model: FV1000)

3. Objective, 10 × 0.3 (Olympus, model: UPLFLN10X2)

4. Objective, 20 × 0.5 (Olympus, model: UPLFLN20X)

5. Water-dipping objective, 40 × 0.8 (Olympus, model: LUMPLFLN40XW)

6. Water-dipping objective, 60 × 1.0 (Olympus, model: LUMPLFLN60XW)

Software and datasets

1. For imaging: FV10-ASW (Olympus, version: 4.1); requires a license

2. For image processing: ImageJ (version: 1.54g, available at https://imagej.net/)

3. For image analysis:

a. Python (version: 3.12, available at https://www.python.org/)

b. Miniconda (available at https://docs.anaconda.com/)

c. napari [12] (available at https://napari.org/)

4. For data analysis:

a. R (version: 4.4.2, available at https://www.cran.r-project.org/)

b. RStudio (version: 2024.09.1, available at https://posit.co/products/open-source/rstudio/)

c. RTools (version: 4.4, available at https://www.cran.r-project.org/)

d. nervecount-napari (version: 0.1.0-beta [13], available at https://github.com/valusty/nervecount-napari)

Note: Software for image analysis and data analysis was used in this article solely for validation purposes on confocal images of transverse sections, not whole-mount preparations.

Procedure