Antibody staining - theunissenlab/lab-documentation GitHub Wiki

Immunohistochemistry protocol (for ChAT antibody but can be adapted for others)

3 days total. Adapted from Feller Lab protocol.

DAY ONE:

Step 0: Perfuse and fix brain in 4% pfa. Then transfer to 30% sucrose for slicing. Once the brain has sunk, this protocol can begin

Step 1: Wash slices 3x in 1xPBS for 5 min each at room temperature

Step 2: Block with 10% Normal Goat Serum (NGS) for 2 hours at room temperature For 10mL of 10% NGS use 1mL of NDS stock (frozen) 9mL of 1x PBS

Step 4: Incubate in primary antibody (anti-GFP) at 1:200 dilution in 0.4% Triton-X and 1% NDS for O/N at 4 degrees For 10mL of 1% NGS + 0.4% Triton-X use 1mL of 10% NGS from previous step 9mL of 1x PBS (or 8.960mL) 40uL of Triton-X

DAY TWO

Step 5: Wash 3x in 1x PBS, 5 min each at room temperature

Step 6: Incubate in secondary antibody(Alexa Fluor 488) at 1:1000 dilution in in 0.4% Triton-X and 1% NGS from step 4 for 2 hours at room temperature

Step 7: Wash 3x in 1x PBS, 5 min each at room temperature

Step 8: mount with vectashield mounting media