Effect of microwave irradiation on antibody labeling efficiency when applied to ultrathin cryosections through fixed biological material.
To study the effect of microwaves on immunolabeling, ultrathin cryosections or diluted antibodies were irradiated prior to antibody application, and gold labeling was quantified. In addition, affinity purified, polyclonal antibodies and protein A-gold were applied to ultrathin cryosections of aldehyde-fixed material in the presence and absence of microwaves. Amylase, a soluble protein secreted by pancreatic acinar cells, MHC class II, an integral membrane protein, and 3-(2,4-dinitroanilino)-3-amino-N-methyldipropylamine (DAMP), an exogenously added antigen, were localized with monospecific antibodies. Each was chosen for their contrasting subcellular characteristics. Results demonstrated that for some antigens, antibody labeling efficiency was quantitatively improved by microwave irradiation of sections prior to antibody application. Irradiation of diluted antibodies prior to their application also resulted in improved labeling. In contrast, the results obtained using rapid immunolabeling protocols in the presence of microwaves resulted in labeling levels similar to those obtained in the absence of microwaves. We conclude that microwave irradiation can improve the labeling efficiency of some antibodies. However, improvements in labeling density are dependent on the antigen under study and on variable irradiation times, unique to each antibody. This suggests that the routine use of microwaves to reduce incubation times may not be a viable alternative to bench protocols.
Center for Cell Imaging, Yale University School of Medicine, Department of Cell Biology, New Haven, Connecticut 06520-8002, USA.
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D000681:Amylases / Q000032:analysis; D000818:Animals; D000906:Antibodies / Q000528:radiation effects; D002460:Cell Line; D016616:Cryoultramicrotomy / Q000379:methods; D004136:Dinitrobenzenes / Q000032:analysis; D006801:Humans; D007089:Image Enhancement; D007150:Immunohistochemistry / Q000379:methods*; D008285:Major Histocompatibility Complex / Q000276:immunology; D008297:Male; D008565:Membrane Proteins / Q000032:analysis; D051379:Mice; D008872:Microwaves; D010179:Pancreas / Q000737:chemistry; D011817:Rabbits; D051381:Rats; D017207:Rats, Sprague-Dawley
D000906:Antibodies; D004136:Dinitrobenzenes; D008565:Membrane Proteins; C042432:3-(2,4-dinitroanilino)-3'-amino-N-methyldipropylamine; D000681:Amylases