Application of Microwave Technology to the Processing and Immunolabeling of Plastic-embedded and Cryosections

Abstract

S U M M A R YWe 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.

Exocytosis sensitivity to growth hormone-releasing hormone in subsets of GH cells in rats under different corticosterone conditions. Ultrastructural study using microwave irradiation for fixation and immunocytochemistry

“…The great increase in molecular motion induced by MWI enables fixatives to soak rapidly and deeply into cells and tissues (Boon et al 1990, Rangell & Keller 2000. In the present study, we applied MWI for fixation and immunocytochemistry using rat GH antibody.…”

“…Some recent papers have indicated preferential immunolabeling density by MWI (Rangell & Keller 2000, Paupard et al 2001. These results were obtained using acrylic resin (such as LR Gold, LR White) or frozen sections.…”

“…The MWI has been widely utilized to advance biological processing: tissue fixation, histochemistry, antigen retrieval, and in situ hybridization (Shi et al 1991, Login et al 1995, Van Ginneken et al 1998, Rangell & Keller 2000, Mitchell et al 2001, Paupard et al 2001. The great increase in molecular motion induced by MWI enables fixatives to soak rapidly and deeply into cells and tissues (Boon et al 1990, Rangell & Keller 2000.…”

“…Some previous studies indicated that post-embedding methods applying microwave irradiation (MWI) to the fixation and immunolabeling of thin plasticembedded sections give good results (Zondervan et al 1988, Login et al 1995, Rangell & Keller 2000. In the present study, we applied microwave technology to processing and immunolabeling of plastic-embedded tissue for the analysis of exocytosis in GH cells stimulated by GRH under different corticosteroid conditions.…”

Morphological and phenotypic changes of nociceptive primary afferent neurons following neurotmesis and perineural capsaicin treatment

“…To generate TRPV1 gene-specific probes, total mRNA isolated from rat trigeminal ganglia was reverse transcribed using the universal until further processing. In order to improve the antigen retrieval sections were heated with microwave irradiation (5 + 4 min in 0.01 M Na-citrate solution, pH 6), left in the same buffer for 10 min until the forthcoming steps (Yang et al, 1999, Rangell and Keller, 2000, Mitchell et al, 2001, Relf et al, 2002, Szigeti et al, 2003. The specimens were fixed for 5 min in 2x sodium chloride -sodium citrate ( The CSA and ROD for each cell were determined and plotted as distribution histograms or scatter plots.…”