Application of microwave technology to the processing and immunolabeling of plastic-embedded and cryosections.
We have adapted existing microwave irradiation (MWI) protocols and applied them to the processing and immunoelectron microscopy of both plastic-embedded and frozen sections. Rat livers were fixed by rapid MW irradiation in a mild fixation solution. Fixed liver tissue was either cryosectioned or dehydrated and embedded in Spurr's, Unicryl, or LR White resin. Frozen sections and sections of acrylic-embedded tissue were immunolabeled in the MW oven with an anti-catalase antibody, followed by gold labeling. Controls were processed conventionally at room temperature (RT). The use of MWI greatly shortened the fixation, processing, and immunolabeling times without compromising the quality of ultrastructural preservation and the specificity of labeling. The higher immunogold labeling intensity was achieved after a 15-min incubation of primary antibody and gold markers under discontinued MWI at 37C. Quantification of the immunolabeling for catalase indicated a density increase of up to fourfold in the sections immunolabeled in the MW oven over that of samples immunolabeled at RT. These studies define the general conditions of fixation and immunolabeling for both acrylic resin-embedded material and frozen sections.
Rangell
L K
LK
Pharmacological Sciences, Genentech, Inc., South San Francisco, California 94080, USA.
Keller
G A
GA
D000180:Acrylic Resins; D000818:Animals; D000906:Antibodies; D002374:Catalase / Q000276:immunology; D016616:Cryoultramicrotomy; D007089:Image Enhancement; D008099:Liver / Q000201:enzymology; D008297:Male; D016253:Microscopy, Immunoelectron / Q000379:methods*; D008872:Microwaves; D016613:Plastic Embedding; D051381:Rats
D000180:Acrylic Resins; D000906:Antibodies; D002374:Catalase