Fixing Tissue & Making Blocks:
1) After tissue has been removed, fix in 4% paraformaldehyde for two hours at 4 C
2)Transfer tissue to PBS overnight at 4 C
3) Transfer tissue to 30% sucrose for 2 hours at 4 C
4) Place tissue in block as desired, fill block with O.C.T. embedding medium and place on dry ice until frozen
5) 4% Paraformaldehyde For 100 mL: 90mL MQ H20 and heat to 60-80 C
6) Measure 4 grams paraformaldehyde reagent and add to water (after it has reached desired temperature)
7) Add 1-2 drops of 10N NaOH-to clear
8) Once clear transfer to ice to cool. After cooled, add 10mL 10X PBS
9) Store at 4C
Staining with Primary Antibody:
1) Wash sections in dH2O three times for 5 minutes each.
2) Wash section in wash buffer for 5 minutes.
3) Block each section with 100-400 ul blocking solution for 1 hour at room temperature.
4) Remove blocking solution and add 100-400 ul primary antibody diluted in recommended antibody diluent to each section. Incubate overnight at 4C.
5) Remove antibody solution and wash sections in wash buffer three times for 5 minutes each.
6) Add 100-400 ul biotinylated secondary antibody, diluted in TBST per manufacturer's recommendation, to each section. Incubate 30 minutes at room temperature.
7) If using ABC avidin/biotin method, prepare ABC reagent according to the manufacturer's instructions and incubate solution for 30 minutes at room temperature.
8) Remove secondary antibody solution and wash sections three times with wash buffer for 5 minutes each.
9) Add 100-400 ul ABC reagent to each section and incubate for 30 minutes at room temperature.
10) Remove ABC reagent and wash sections three times in wash buffer for 5 minutes each.
11) Add 100-400 ul DAB or suitable substrate to each section and monitor staining closely.
12) As soon as the sections develop, immerse slides in dH2O.
13) If desired, counterstain sections in hematoxylin per manufacturer's instructions.
14) Wash sections in dH2O two times for 5 minutes each.
15) Dehydrate sections: Incubate sections in 95% ethanol two times for 10 seconds each.
16) Repeat in 100% ethanol, incubating sections two times for 10 seconds each.
17) Repeat in xylene, incubating sections two times for 10 seconds each.
18) Mount coverslips.
Secondary Antibody Staining:
1) Wash slides with PBT 6 x 5
2) Add 500 uL of diluted secondary antibody, in blocking buffer, to each slide
3) Cover and incubate at room temp for 1 hour and 30 minutes
4) Wash slides with PBT 6 x 5
5) Mount slides w/ Vectashield