Pieri L, Sassoli C, Romagnoli P, Domenici L
Dipartimento di Anatomia, Istologia e Medicina Legale, University of Florence, Florence, Italy.
Eur J Histochem. 2002;46(4):365-75. doi: 10.4081/1749.
Periodate-lysine-paraformaldehyde (PLP) has been proposed as a fixative for glycoprotein antigens which should stabilize periodate oxidized polysaccharide chains through lysine mediated crosslinks, either directly or by the intermediation of formaldehyde. In spite of premises and attempts reported in the literature, this fixative has never become popular for the study of membrane antigens of immune system cells, which leads to doubts on its real efficacy. We have addressed this issue in biopsies of human skin and found that PLP followed by cryoprotection with 30% sucrose and cryosectioning, or PLP fixation of isolated epidermal sheets, consistently provided for good preservation of morphology and intense labeling of major histocompatibility complex class II molecules, CD 1 a, CD4, CD8, E-cadherin, cytokeratins in general, cytokeratin-18 in particular, and bromodeoxyuridine, incorporated by cycling cells in vitro, and for the demonstration of tyrosinase enzyme activity. PLP-fixed, osmicated and epon-embedded epidermal sheets proved as good as sheets fixed with a mixture of formaldehyde and glutaraldehyde for electron microscopic morphological analysis. Also, these sheets were amenable to immunoperoxidase staining of Langerhans cell membrane antigen CD1a and keratinocyte membrane antigen E-cadherin before being osmicated and prepared for electron microscopy. In a parallel paper, we had also shown that oral mucosa biopsies fixed in PLP showed good morphology and immunolabeling of CD54, CD80, CD83 and CD86. Therefore, we conclude that PLP can be proposed as a multi-task fixative for light and electron microscopic analysis of membrane, cytoplasmic and nuclear antigens of immune system cells and keratinocytes.
高碘酸盐-赖氨酸-多聚甲醛（PLP）已被提议作为一种用于糖蛋白抗原的固定剂，它应通过赖氨酸介导的交联直接或通过甲醛的介导来稳定高碘酸盐氧化的多糖链。尽管文献中有相关的前提和尝试报道，但这种固定剂在免疫系统细胞膜抗原研究中从未流行起来，这引发了对其实际效果的怀疑。我们在人体皮肤活检中解决了这个问题，发现先用PLP处理，然后用30%蔗糖进行冷冻保护并进行冷冻切片，或者对分离的表皮片进行PLP固定，始终能很好地保存形态，并能强烈标记主要组织相容性复合体II类分子、CD1a、CD4、CD8、E-钙黏蛋白、一般的细胞角蛋白，特别是细胞角蛋白-18，以及体外循环细胞掺入的溴脱氧尿苷，还能用于酪氨酸酶活性的检测。经PLP固定、锇酸处理并嵌入环氧树脂的表皮片，在电子显微镜形态分析方面与用甲醛和戊二醛混合物固定的表皮片一样好。此外，在进行锇酸处理和电子显微镜样品制备之前，这些表皮片适合进行朗格汉斯细胞膜抗原CD1a和角质形成细胞膜抗原E-钙黏蛋白的免疫过氧化物酶染色。在另一篇相关论文中，我们还表明，用PLP固定的口腔黏膜活检标本显示出良好的形态以及CD54、CD80、CD83和CD86的免疫标记。因此，我们得出结论，PLP可被提议作为一种多用途固定剂，用于对免疫系统细胞和角质形成细胞的膜、细胞质和核抗原进行光镜和电镜分析。
