Immunohistochemistry and imaging

[Day 1]

1. Perfuse mouse with ~20 mL 1X PBS and then ~20 mL 4% PFA

2. Dissect out brain

3. Place the brain in scintillation vial of 4% PFA, leave overnight in 4^oC fridge 
[Day 2]

4. Embed brains in 4% agarose in H₂O

5. Use glue to mount brain onto vibratome chamber platform. Fill chamber with PBS

6. Cut 50 micrometer thick slices

7. Collect slices with a paint brush and enter into wells of 24 well plate containing PBS

8. Prepare blocking solution (0.1% Triton X-100 PBS + 4% normalized goat serum, NGS)

9. Replace the PBS in the wells housing the cells with the blocking solution (PBST+NGS) to cover the slices

10. Let sit / block for 1 hour

11. Prepare the primary antibody solution in PBST+NGS using the appropriate antibody dilution. Calculate such that 300 uL of PBST+NGS are in each well. Primary antibodies used were rabbit polyclonal anti-Doc2b (1:200; Synaptic Systems, Cat#174) and mouse monoclonal anti-Parvalbumin (1:500, Sigma-Aldrich, Product #P3088-.2ML)

12. Once done blocking, remove the PBST+NGS from the wells and add 300 uL of the primary antibody in PBST+NGS solution to each appropriate well

13. Incubate with the primary antibody + PBST + NGS soltuion overnight at 4^oC 
[Day 3]

14. Wash slices 3 x 10 min with PBST+NGS

15. Meanwhile, prepare secondary antibodies with appropriate dilution once again using PBST+NGS.Secondary antibodies used were goat anti-rabbit IgG H and L Alexa Fluor647 (1:1000, ab150083) and goat anti-mouse IgG H and L Alexa Fluor568 (1:1000, ab175473)

16. Incubate slices with the secondaries in the dark, 1-2 hours at RT.

17. Wash 3 x 5 min in PBS (not PBS!)

18. Mount slices on glass slides and cover with mounting medium (Invitrogen Fluoromount-G Mounting Medium, Cat#00-4958-02) and glass coverslip

19. Let mounting medium cure for at least 24 hours before imaging

20. Image whole brain slices on Olympus VS120 slide scanner or Olympus FV1000 confocal microscopes collecting 10 micrometer z-stacks

Solutions

PBST: 0.1% Triton X-100 PBS

PBST-NGS (blocking buffer): 0.1% Triton X-100 PBS + 4% normalized goat serum

Tested primary antibodies

Rabbit polyclonal anti-Doc2b (1:200), Synaptic Systems, Cat#174

Mouse monoclonal anti-Parvalbumin (1:500), Sigma-Aldrich, Product #P3088-.2ML

Category