SIMPLE FLUORESCENCE METHOD FOR SEROTONIN-CONTAINING ENDOCRINE-CELLS IN PLASTIC-EMBEDDED LUNG, GUT AND THYROID-GLAND

Small-granule, presumptively endocrine cells in the lungs are difficult to identify by routine light microscopy, and demonstration of serotonin (5-HT) and dopamine in these cells after Falck and Owman using lyophilization and exposure to hot formaldehyde gas at controlled humidity, is difficult, time consuming and unsuited to large scale histologic surveys. The authors report fluorescence localization of a variety of 5-HT-containing APUD endocrine cells, obtained without resort to freeze drying or formaldehyde gas. Tissue is fixed at room temperature by intravascular perfusion with or immersion in 6% w/v aqueous, phosphate buffered (0.1 M, pH 7.2) formaldehyde, freshly prepared from paraformaldehyde powder. Samples can be left in fixative for days or weeks before rinsing in H2O, dehydrating in graded ethanols and embedding in glycol methacrylate or paraffin. Sections are cut, floated with H2O onto glass slides, air dried (paraffin sections are dewaxed in xylene), mounted in Entellan and examined with an epifluorescence microscope. Strong yellow fluorescence emanates from cells known to contain 5-HT (enterochromaffin cells of the gut, mast cells of rats, small-granule cells of rabbit lungs) and from APUD cells in the thyroid glands and tracheal epithelum of rats, if the latter are preinjected with the 5-HT precursor, 5-hydroxytryptophan (5-HTP). Yellow fluorescence is abolished by treatment of sections with sodium borohydride; it is not reestablished by exposing plastic sections to formaldehyde gas at 80°C but does reappear in dewaxed paraffin sections. The method works only for 5-HT; it does not demonstrate catecholamines in adrenal medulla or dopamine in thyroid glands or tracheal epithelium of rats preinjected with the precursor, L-DOPA. It affords excellent resolution of cellular detail in 2 μm plastic sections and is well suited for work requiring serial reconstructions, in as much as slides are reliably obtained and fluorescence can be demonstrated in them weeks or months later.