Materials and reagents

1. High-profile microtome blades (Leica, catalog number: 14035838383)

2. Glass insert 70 mm (Leica, catalog number: 14047742497)

3. Micro slides (Muto Pure Chemicals, catalog number: GA13-7626R)

4. O.C.T (VWR, catalog number: 95057838)

5. Aluminum foil (Kirkland, catalog number: RK611)

6. 10× phosphate-buffered saline (PBS) (Omics Bio, catalog number: IB3012)

7. Sucrose (Sigma-Aldrich, catalog number: S0389-500G)

8. Glycine, powder (Omics Bio, catalog number: BT5031)

9. Paraformaldehyde powder (Sigma-Aldrich, catalog number: P6148-500G)

10. Alexa-594-conjugated α-bungarotoxin (Life Technologies, catalog number: B13423)

11. DAPI fluoromount-G (SouthernBiotech, catalog number: 0100-20)

12. Microscope cover glass (Shinetech Inc, catalog number: GA11-2450)

13. 1.5 mL clear microcentrifuge tubes (Corning, catalog number: MCT-150-C)

14. 2,2,2-tribromoethanol (avertin) (Sigma-Aldrich, catalog number: T48402-5G)

15. Horse serum (Gibco, catalog number: 16050-114)

16. BSA albumin fraction V (BioFroxx, catalog number: 4240GR100)

17. Anti-synaptophysin primary antibody (Abcam, catalog number: ab32127)

18. Anti-neurofilament primary antibody (Abcam, catalog number: ab8138)

19. 488-conjugated donkey anti-rabbit secondary antibody (Jackson ImmunoResearch, catalog number: AB_2340620)

1. 1× PBS (see Recipes)

2. 4% PFA (see Recipes)

3. 2.5% avertin (see Recipes)

4. 0.1 M glycine solution (see Recipes)

5. 30% sucrose (see Recipes)

6. Blocking buffer (see Recipes)

1. 1× PBS

   Reagent	Final concentration	Amount
   10× PBS	n/a	200 mL
   Deionized H2O	n/a	1800 mL
   Total	n/a	2000 mL

   Note: The 1× PBS solution should be prepared fresh and used within seven days.




2. 4% PFA

   Reagent	Final concentration	Amount
   Paraformaldehyde powder	4%	4 g
   1× PBS	n/a	100 mL
   Total	n/a	100 mL

   Note: To prepare 4% PFA solution, add 4 g of paraformaldehyde powder to 90 mL of 1× PBS, stir, and heat to 60–65 °C until dissolved. Add a few drops of 5 N NaOH to clear the solution (5 N NaOH is used to facilitate the dissolution of paraformaldehyde powder), cool to room temperature, add 1× PBS to 100 mL, and adjust the pH to 7.4. The solution can be prepared in advance and stored in a -20 °C freezer for long-term storage. Thaw the solution 30 min before use to ensure it is fully liquefied.




3. 2.5% avertin

   Reagent	Final concentration	Amount
   2,2,2-tribromoethanol	2.5%	250 μL
   1× PBS	n/a	9.75 mL
   Total	n/a	10 mL

   Note: The 2.5% avertin solution can be prepared in advance and stored in a 4 °C refrigerator for up to one month.




4. 0.1 M Glycine solution

   Reagent	Final concentration	Amount
   Glycine powder	0.1 M	0.75 g
   1× PBS	n/a	10 mL
   Total	n/a	10 mL

   Note: To prepare a 0.1 M glycine solution, add 0.75 g of glycine to 10 mL of 1× PBS in a 15 mL conical centrifuge tube and shake until the solution is clear. There is no need to adjust the pH of the solution. The solution can be prepared in advance and stored in a 4 °C refrigerator for up to two months.




5. 30% sucrose

   Reagent	Final concentration	Amount
   Sucrose	30%	15 g
   1× PBS	n/a	50 mL
   Total	n/a	50 mL

   Note: To prepare a 30% sucrose solution, add 15 g of sucrose to 50 mL of 1× PBS and shake until the solution is clear. The solution can be prepared in advance and stored in a 4 °C refrigerator for up to one month.




6. Blocking buffer

   Reagent	Final concentration	Amount
   BSA albumin fraction V	2%	0.2 g
   1× PBS	n/a	9.5 mL
   Horse serum	5%	500 μL
   Total	n/a	10 mL

   Note: To prepare the blocking buffer, add 500 μL of horse serum to 9.5 mL of 1× PBS in a 15 mL conical centrifuge tube, shake, and then add 0.2 g of BSA albumin fraction V into the solution. Shake until the solution is clear.

Equipment

1. P2, P200 and P1000 micropipettes

2. Laser scanning confocal microscope (Zeiss, model: LSM880)

3. Staining jar (Shineteh Instruments, catalog number: GB11-15)

4. Slide racks (Shineteh Instruments, catalog number: GB11-15S)

5. Liquid nitrogen insulation barrels (Shineteh Instruments, catalog number: IE1-D6000)

6. Cryostat (Leica, catalog number: CM3050)

7. 4 °C, -20 °C freezers [Sampo, catalog number: SR-C61G(K3)]

8. -80 °C freezers (Panasonic, catalog number: MDF-U74V)

9. Laminar flow cabinet (CHUAN, catalog number: TBH-520M)

10. Orbital shaker (TKS, catalog number: OS-701)

11. Disposable syringe with needle (Terumo, catalog number: MDSS01S2613)

12. Disposable syringe (Terumo, catalog number: MDSS20ES)

13. Iris scissors (Shineteh Instruments, catalog number: ST-S009)

14. Fine curve point tweezers (Shineteh Instruments, catalog number: ST-NO7)

15. Super fine point tweezers (Shineteh Instruments, catalog number: ST-NO3)

16. Iris forceps (Shineteh Instruments, catalog number: ST-I210)

17. Lab mat (Dogger, catalog number: D4EG-031055)

18. Falcon^TM 15 mL conical centrifuge tubes (Thermo Fisher, catalog number: 14-959-53A)

19. Immuno-pathology staining kit (PEOPLE LIFE TEK CO., LTD., catalog number: H198099910)

Software and datasets

1. ImageJ image processing and analysis software (https://imagej.net/ij/)

2. ZEN microscopy software (https://www.zeiss.com/microscopy/en/products/software/zeiss-zen.html)

3. GraphPad Prism 8.0.2

Procedure