A simple fluorescence method for serotonin-containing endocrine cells in plastic-embedded lung, gut and thyroid gland.
A simple fluorescence method for serotonin-containing endocrine cells in plastic-embedded lung, gut and thyroid gland.
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一种简单的荧光方法，用于检测塑料包埋的肺、肠和甲状腺中含血清素的内分泌细胞。
DOI：
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发表时间：
1979
影响因子：
3.2
通讯作者：
R. Bartlett
中科院分区：
文献类型：
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作者：
R. Hoyt;S. Sorokin;R. Bartlett
肺中的小颗粒、假定的内分泌细胞难以通过常规光学显微镜鉴定，Faick和Owman（Acta Univ隆德II 7：1（Suppl）1965（6））使用冻干和暴露于受控湿度下的热甲醛气体后，证明这些细胞中的5-羟色胺（5-LIT）和多巴胺是困难、耗时且不适合大规模组织学研究的。我们报道了多种含5-HT的APIJD内分泌细胞的荧光定位（Journal of Histochemistry and Cytochemistry 17：303，1969（11）），这些细胞是在不采用冷冻干燥或甲醛气体的情况下获得的。在室温下，通过血管内灌注或浸入6%w/v磷酸盐缓冲甲醛水溶液（0.1 M，pH 7.2）（由多聚甲醛粉末新鲜制备）固定组织。样品可以在固定剂中放置数天或数周，然后在H2O中冲洗，在分级乙醇中脱水并包埋在乙二醇甲基丙烯酸酯或石蜡中。切片，用H2O漂浮在载玻片上，风干（石蜡切片在二甲苯中脱蜡），在Entellan（E.M.实验室有限公司Elmsford，N.Y.）并用落射荧光显微镜检查。强黄色荧光来自已知含有5-HT的细胞（肠嗜铬细胞、大鼠肥大细胞、兔肺小颗粒细胞）以及大鼠甲状腺和气管上皮中的APUD细胞，如果后者预先注射了5-HT前体5-羟色氨酸（5-HTP）。用硼氢化钠处理切片可消除黄色荧光;将塑料切片暴露于80 ℃的甲醛气体中不能重新产生黄色荧光，但在脱蜡石蜡切片中会重新出现。该方法仅适用于5-HT;它不能证明预先注射前体L-DOPA的大鼠肾上腺髓质中的儿茶酚胺或甲状腺或气管上皮中的多巴胺。它在2 pm塑料切片中提供了细胞细节的出色分辨率，非常适合需要连续重建的工作，因为可以可靠地获得载玻片，并且可以在几周或几个月后在其中显示荧光。
Small-granule, presumptively endocrine cells in the lungs are difficult to identify by routine light microscopy, and demonstration ofserotonin (5-LIT) and dopamine in these cells after Faick and Owman (Acta Univ Lund II 7:1 (Suppl) 1965 (6)), using lyophilization and exposure to hot formaldehyde gas at controlled humidity, is difficult, time consuming and unsuited to large scale histologic surveys. We report fluorescence localization of a variety of 5-HT-containing APIJD (Journal of Histochemistry and Cytochemistry 17:303, 1969 (11)) 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 H20, dehydrating in graded ethanols and embedding in glycol methacrylate or paraffin. Sections are cut, floated with H20 onto glass slides, air dried (paraffin sections are dewaxed in xylene), mounted in Entellan (E.M. Laboratories, Inc., Elmsford, N.Y.) 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 epithelium 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#{176}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 pm 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.