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Anesthetize the mouse/rat and perfuse transcardially with 3-6 ml 0.1M phosphate buffer (PB, pH 7.3) with 1000 units/ml of heparin, respectively, followed by 1% glutaraldehyde/ 0.5 % paraformaldehyde/0.1% picric acid in 0.1M PB. Fix brain tissue overnight in the cold then wash with PB.
-
Cut 60-70μm sections on a vibratome and place into cold 0.1M PB.
-
Place sections into well plate and continue with microwave pre-embed immune protocol below
*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 Retrieval |
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 |
1 |
Cont |
| 7 |
Dilute Blocking Rinse |
0:01:00 |
150 |
35 |
2 |
Off |
| 8 |
Secondary Ab |
0:15:20² |
200 |
35 |
1 |
Cont |
| 9 |
Imidazole Rinse |
0:01:00 |
150 |
35 |
2 |
Off |
| 10 |
ABC in Imidazole |
0:15:20² |
150 |
35 |
1 |
Cont |
| 11 |
Imidazole Buffer Rinse |
0:01:00 |
150 |
35 |
2 |
Off |
| 12 |
DAB |
0:05:20³ |
200 |
35 |
1 |
Cont |
| 13 |
Imidazole Buffer Rinse |
0:01:00 |
150 |
35 |
2 |
Off |
| 14 |
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.
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