RAPID PRIMARY MICROWAVE-ALDEHYDE AND MICROWAVE-OSMIUM FIXATION - IMPROVED DETECTION OF RAT PAROTID ACINAR CELL SECRETORY GRANULE ALPHA-AMYLASE USING A POSTEMBEDDING IMMUNOGOLD ULTRASTRUCTURAL MORPHOMETRIC ANALYSIS

Studies of methods for improved fixation ace becoming increasingly imp ortant in the field of quantitative immunocytochemistry. We used micro wave (MW)-assisted chemical fixation to show improved retention of sal ivary gland acinar cell secretory granule alpha-amylase detected by a quantitative immunogold method. Blocks (4-mm(3)) of rat parotid gland were fixed by the following methods: (a) MW irradiation in an aldehyde fixative (AL) for 6 sec; (b) immersion in AF for 1.5 hr; (c) MW irrad iation in osmium tetroxide (OT) for 9 sec; (d) immersion in OT for 1.5 hr; or (e) Sequential MW AF, 10 sec, MW OT rapid treatment (SMAORT), 10 sec. Specimens were processed routinely for transmission electron m icroscopy. Thin sections of Epon-embedded tissues were exposed first t o rabbit IgG anti-human salivary alpha-amylase and second to gold-conj ugated goat anti-rabbit IgG. Granule area was obtained by a point coun ting method. Labeling density was calculated as the number of gold par ticles/mu m(2) +/- SD. Specimens fixed in seconds by MW-AE, MW-OT, or SMAORT showed ultrastructral preservation similar to immersion fixatio n in AF or OT for 1.5 hr. Immunogold labeling density of granule alpha -amylase was highest for SMAORT (874 mu m(2)) compared to MW-AF (295 m u m(2)), MW-OT (248 mu m(2)), routine sequential immersion in AF and O T (229 mu m(2)), or immersion in CYT (no aldehyde) (190 mu m(2)). This study establishes the improved retention of salivary gland acinar cel l secretory granule a-amylase and markedly enhanced fixation speed for ultrastructural studies made possible by MW-chemical ft ration protoc ols that use aldehydes and osmium.