Fixing Tissue & Making Blocks:
 

• After tissue has been removed, fix in 4% paraformaldehyde for two hours at 4°C
• Transfer tissue to PBS overnight at 4°C
• Transfer tissue to 30% sucrose for 2 hours at 4°C
• Place tissue in block as desired, fill block with O.C.T./ embedding medium and place on dry ice until frozen

• For 100 mL:  90mL MQ H20 and heat to 60-80*
• Measure 4 grams paraformaldehyde reagent and add to water (after it has reached desired temperature)
• Add 1-2 drops of 10N NaOH-to clear
• Once clear transfer to ice to cool
• After cooled, add 10mL 10X PBS
• Store at 4°C

    Staining with Primary Antibody:

• Wash slides with PBT (Phosphate Buffer Saline with 0.02%Tween 20)  3  x  5’ (3 times, 5 minutes each)
• Add 500µL of diluted antibody, in blocking buffer**, to each slide
• Cover and store slides at 4°C overnight OR incubate at room temperature for 2 hours (Depending on the antibody)

    Secondary Antibody Staining:
 

• Wash slides with PBT 6 x 5’
• Add 500 µL of diluted secondary antibody, in blocking buffer**, to each slide
• Cover and incubate at room temp for 1 hour and 30 minutes
• Wash slides with PBT 6 x 5’
• Mount slides w/ Vectashield

    **Blocking Buffer:

    (When using a goat antibody, do not add HINGS)

        For 40 ml :

•   40 ml PBT
•   400µl HINGS (heat inactivated goat serum)
•   400µl HINDS (heat inactivated donkey serum)
•   400µl 10% Triton X-100