Immunohistochemistry

IHC protocol with anti-BG4 for brain floating sections

Mice were transcardially perfused with cold PBS and then with cold 4% paraformaldehyde/PBS. Brains were isolated and immersed in 30% sucrose/PBS for 3 days at 4°C for dehydratation.

Brains were sectioned with a microtome (25 um thickness) and stored with anti-freeze solution in 96-well plates at -20°C.

Antifreeze solution recipe: 300 mL Ethyleneglycol

300 mL Glycerol

300 mL distilled H2O

100 mL 2x PO4 Buffer (0.244 M)

2x PO4 Buffer (0.244 M) solution recipe:

Immunohistochemistry

1. Transfer brain slices with a fine paintbrush in to a 96-well plate with 150 µl blocking buffer (5% BSA + 0.5% Triton-X in PBS). Up to two brain slices perwell.
2. Incubate overnight (ON) at 4°C
3. Transfer brain slices in to wells with primary antibody solution (100 µl/well):
   1. Anti-BG4 (produced by Dr. Kim lab), 2 µg/100 µl in blocking buffer
4. Incubate ON at 4°C
5. Transfer brain samples in to a 24-well plate with PBS + 1% Tween (PBS-T) (1 mL/well) for washing
6. Wash 3 times for 5 m each wash, by aspirating PBS-T and adding 1 mL of PBS-T after each wash
7. Transfer brain slices in to a 96-well plate for incubation with anti-FLAG
   1. Anti-FLAG (#2368, Cell Signaling), 1:100 in blocking buffer
8. Incubate at RT for 1 h
9. Transfer brain slices in to a 24-well plate with PBS + 1% Tween (PBS-T) (1 mL/well) for washing
10. Wash for 5 m 3 times by aspirating PBS-T and adding 1 mL of PBS-T after each wash
11. Transfer brain slices in to a 96-well plate for incubation with secondary antibody solution:
    1. Anti-rabbit-488 (abcam, ab150077), 1:500 in blocking buffer
12. Incubate at RT for 1 h
13. Transfer brain slices in to a 24-well plate with PBS + 1% Tween (PBS-T) (1 mL/well) for washing
14. Wash for 5 m 3 times by aspirating PBS-T and adding 1 mL of PBS-T after each wash
15. Add 0.1 µg/mL Hoechst solution in PBS (500 µl/well) and incubate for 2 m at RT
16. Wash for 5 m 3 times by aspirating PBS-T and adding 1 mL of PBS-T after each wash
17. Add 1x True-black (enough to cover samples) and incubate for 30 s – 1 m
18. Wash for 5 m 3 times by aspirating PBS-T and adding 1 mL of PBS-T after each wash
19. Mount brain samples on glass slides
20. Add Vectashield antifade mounting media (VectorLabs) and cover samples with aglass coverslip
21. Keep in darkness at 4°C until imaging