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PELCO BioWave® Pro+ Microwave Tissue Processor
Electron Microscopy Tissue Processing Application Kit

Microwave Pre-embedding Labeling (DAB)

Protocol for Electron Microscopy

Microwave Procedure by CHARLES K. MESHUL, PH.D.
Professor, Department of Behavioral Neuroscience and Pathology (OSHU)
Research Biologist and Director, Electron Microscopy Facility (VA)

  1. 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.
  2. Cut 60-70μm sections on a vibratome and place into cold 0.1M PB.
  3. 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.