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go to Electron Microscopy Tissue Processing Applicaiton Kit
Microwave Procedure by CHARLES K. MESHUL, PH.D.
Professor, Department of Behavioral Neuroscience and Pathology (OSHU)
Research Biologist and Director, Electron Microscopy Facility (VA)
*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 | 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.