*note: for the procedure below in the microwave, use just enough liquid to cover tissue, approx. 100ul/well
| Steps# |
Description |
Time
(hr:min:sec) |
Watts |
Temperature |
Repetitions |
Vacuum |
| 1 |
Antigen Retreival |
0:05:00 |
550 |
35 |
1 |
Cycle |
| 2 |
PBS Rinse |
0:01:00 |
150 |
35 |
1 |
Off |
| 3 |
0.3% Hydrogen Peroxide |
0:01:00 |
150 |
35 |
1 |
Off |
| 4 |
PBS Rinse |
0:01:00 |
150 |
35 |
3 |
Off |
| 5 |
0.5 Triton x 100 |
0:05:00 |
550 |
35 |
1 |
Cycle |
| 6 |
Primary Ab |
0:12:20* |
200 |
35 |
4 |
Cont |
| 7 |
Dilute Blocking Rinse |
0:01:00 |
150 |
35 |
2 |
Off |
| 8 |
Secondary Ab |
0:15:20² |
200 |
35 |
4 |
Cont |
| 9 |
PBS Rinse |
0:01:00 |
150 |
35 |
2 |
Off |
* =10 SEC PREVAC(CYCLE)/ 2 MIN WATTS ON(CONT VAC)/2 MIN WATTS OFF(CONT VAC)/2 MIN WATTS ON(CONT VAC)/5 MIN WATTS OFF(CONT VAC)/10 SEC FINISH(NO VAC)
² = 10 SEC PREVAC(CYCLE)/ 4 MIN WATTS ON(CONT VAC)/3 MIN WATTS OFF(CONT VAC)/4 MIN WATTS ON(CONT VAC)/5 MIN WATTS OFF(CONT VAC) /10 SEC FINISH(NO VAC)
³ = 10 SEC PREVAC(CYCLE)/5 MIN WATTS ON /10 SEC FINISH(NO VAC)
Solutions:
Antigen retrieval:10mM sodium citrate 6.0 or 10mM Tris-1mM EDTA 9.0, which is antibody dependent. This will need to be customized for each antibody.
Dilute Blocking solution: 1% Normal goat serum; 1% BSA;0.3% TWEEN 20
Secondary: biotinylated anti-mouse, -rabbit, -rat, -goat, etc: 1:50 dilution
ABC: ABC Elite Kit from Vector; 1ul/ml of each solution A and B in PBS
Imidazole Buffer: 75.5mls of Millipore filtered deionized water; 17.5mls of 1M NaAc pH 7.2; 5mls of 0.2M imidazole
DAB: 0.5ug/ml+1.5% hydrogen peroxidel.