Greenlee J E, Keeney P M
Stain Technol. 1982 Jul;57(4):197-205. doi: 10.3109/10520298209066709.
Trypsin and protease V (pronase) were studied for their ability to enhance immunofluorescent labelling of papovavirus antigens in glycol methacrylate embedded sections of organs infected with murine K-papovavirus. Treatment of Bouin's fixed sections with 0.4% trypsin for 30 minutes resulted in specific immunofluorescent staining equal to that seen in frozen sections and produced little if any loss of histological detail. Treatment with protease V resulted in less brilliant fluorescence and less satisfactory tissue preservation. Studies were then conducted to determine the fluorescence and less satisfactory tissue preservation. Studies were then conducted to determine the fixative which would produce brightest specific fluorescent antibody staining of papovavirus-infected cells while providing clearest definition of intranuclear inclusions and best morphological detail in histologically stained adjacent sections. Brightest immunofluorescence staining was accomplished on material fixed in 96% ethanol/1% glacial acetic acid or Bouin's solution. These fixatives also gave clear definition of intranuclear inclusions with histological stains and provided excellent morphological detail. Phosphate buffered paraformaldehyde/picric acid and 3.7% formalin gave less satisfactory fluorescence and obscured intranuclear inclusions in histological preparations. Sections fixed in 4% paraformaldehyde, 4% paraformaldehyde/1% glutaraldehyde, and 0.5 M p-toluenesulfonic acid were negative for specific fluorescence. Glycol methacrylate, used with proper fixation and trypsin pretreatment of sections, provides a useful embedding medium for immunofluorescent identification of virus-infected cells, and the 1.0-2.0 micron sections routinely obtainable with GMA permit study of individual infected cells by fluorescent antibody and histological staining of adjacent sections.
研究了胰蛋白酶和蛋白酶V（链霉蛋白酶）增强免疫荧光标记小鼠K-乳头多瘤空泡病毒感染器官的乙二醇甲基丙烯酸酯包埋切片中乳头多瘤空泡病毒抗原的能力。用0.4%胰蛋白酶处理Bouin固定切片30分钟，可产生与冷冻切片相当的特异性免疫荧光染色，且组织学细节几乎没有损失。用蛋白酶V处理则导致荧光较弱且组织保存效果较差。随后进行研究以确定能在对乳头多瘤空泡病毒感染细胞产生最亮特异性荧光抗体染色的同时，在组织学染色的相邻切片中提供最清晰的核内包涵体定义和最佳形态细节的固定剂。在96%乙醇/1%冰醋酸或Bouin溶液中固定的材料上实现了最亮的免疫荧光染色。这些固定剂在组织学染色中也能清晰显示核内包涵体，并提供了出色的形态细节。磷酸盐缓冲多聚甲醛/苦味酸和3.7%福尔马林的荧光效果较差，且在组织学制剂中会掩盖核内包涵体。在4%多聚甲醛、4%多聚甲醛/1%戊二醛和0.5M对甲苯磺酸中固定的切片特异性荧光呈阴性。乙二醇甲基丙烯酸酯与适当的固定和切片的胰蛋白酶预处理一起使用，为免疫荧光鉴定病毒感染细胞提供了一种有用的包埋介质，并且用GMA常规可获得的1.0 - 2.0微米切片允许通过荧光抗体研究单个感染细胞以及对相邻切片进行组织学染色。
