CONFOCAL MICROSCOPY OF THICK SECTIONS FROM ACRYLAMIDE-GEL EMBEDDED EMBRYOS
CONFOCAL MICROSCOPY OF THICK SECTIONS FROM ACRYLAMIDE-GEL EMBEDDED EMBRYOS
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DOI：
10.1002/jemt.1070300608
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发表时间：
1995-04-15
影响因子：
2.5
通讯作者：
THOMPSON, RP
中科院分区：
文献类型：
--
作者：
GERMROTH, PG;GOURDIE, RG;THOMPSON, RP
制备光学清晰，厚切片的脆弱的胚胎组织大大有助于共聚焦扫描激光显微镜成像的三维结构的权力。我们在这里报告的条件，包埋，切片，染色胚胎在聚丙烯酰胺凝胶的各种共聚焦成像技术。在10-15%丙烯酰胺单体的标准混合物中渗透组织，聚合后产生易于用振动切片机切割的50 - 1，000 μ m的块。这些条件对于先前染色的组织或对于用低分子量水溶性荧光染料（MW < 5 kD [例如，碘化丙啶，鬼笔环肽]）。对于包埋和切片后组织的免疫染色，将丙烯酰胺浓度降低至2-3%丙烯酰胺，以允许免疫球蛋白进入抗原位点;此类凝胶补充有1%琼脂糖，以便于切片和处理。这两种方法都产生了丰富的，光学清晰，易于处理的部分安装和检查在水溶性介质。(C)1995年Wiley-Liss，Inc.
The preparation of optically clear, thick sections of fragile embryonic tissues greatly aids the power of confocal scanning laser microscopy in imaging three-dimensional structures. We report here conditions for embedding, sectioning, and staining embryos in polyacrylamide gels for a variety of confocal imaging techniques. Infiltration of tissues in standard mixtures of 10-15% acrylamide monomer yields, upon polymerization, blocks that cut easily by vibratome between 50 and 1,000 mu m. These conditions worked well for tissues previously stained or for staining gel sections with low molecular weight water-soluble fluorochromes (MW < 5 kD [e.g., propidium iodide, phalloidin]). For immunostaining of tissue after embedding and sectioning, the acrylamide concentration was reduced to 2-3% acrylamide to allow access of immunoglobulins to antigenic sites; such gels were supplemented with 1% agarose to facilitate sectioning and handling. Either method yielded abundant, optically clear, and easily handled sections for mounting and examination in water-miscible media. (C) 1995 Wiley-Liss, Inc.