Freeze-substitution and postembedding immunocytochemistry on rat taste buds: G-proteins, calcitonin gene-related peptide, and choline acetyl transferase
We have explored freeze-substitution combined with low-temperature embedding in rat taste buds for postembedding immunocytochemistry. A major difference in taste bud cells that were rapidly frozen without prior chemical fixation and those that were fixed and cryoprotected before freezing was that electrondense core granules were virtually absent. The antibodies used in these initial studies were directed against calcitonin gene-related peptide (CGRP), a peptide commonly found in nociceptive neurons; the α-subunits of two G-proteins involved in bitter and sweet taste transduction; and choline acetyl transferase (ChAT), an enzyme involved in the synthesis of acetylcholine. Anti-CGRP immunolabeled a subpopulation of unmyelinated perigemmal neurons; anti-Gqα labeled a larger subpopulation of these neurons and the microvilli of cells that were most likely from Type II vallate taste buds. α-Gustducin was found in cytoplasm of Type II and/or III cells and probably in microvilli of Type I cells of vallate taste buds. The best labeling results were obtained with anti-ChAT, which stained microvilli and lateral membranes of some Type II vallate taste bud cells, and the cytoplasm of some other Type II and/or III vallate cells. In addition, anti-ChAT labeled electron-opaque materials inside taste bud pores of vallate papillae, but, under the same conditions, not granules of Type I cells or most of the vesicles in von Ebner's glands. These data suggest that we can not assume a priori that the contents of the electron-dense core granules of Type I cells, or even of those of von Ebner's glands, contain the precursors of the taste bud pore-dense substances.
Menco, BPM; Yankova, MP; Simon, SA
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