Brandtzaeg P, Rognum T O
Histochem J. 1983 Jul;15(7):655-89. doi: 10.1007/BF01002987.
Eight cross-linking fixatives were tested for preservation of extracellular or intracellular IgG, IgA, IgM, IgD, kappa and lambda light chains, J chain and secretory component. Most of the selected fixatives have been used in recent immunohistochemical studies of lymphoproliferative processes and comprised routine formalin, glutaraldehyde(1%)-formalin, Baker's formalin-calcium, formalin-sublimate, acetic acid(2%)-formalin-saline, Bouin's fluid, Susa fixative, and carbodiimide. The results obtained in artificial test substrates with defined amounts of IgG or IgA and in biological substrates (colon mucosa, tonsils, and different types of lymphomas) were compared by immunofluorescence with the antigenic preservation afforded by fixation in cold 96% ethanol (with or without inclusion of a pre-fixation 48 h washing period). An antigen concentration at least an eight-fold higher was necessary for detection with most other fixatives. Bouin's and Susa fixatives were peculiar in that they required antigen concentration 150 times higher for detection of IgG but only 3-8 times higher for IgA. Light chains were relatively well preserved by all fixatives except glutaraldehyde. For all cross-linking fixatives, the extent of antigenic masking depended on the concentration of environmental proteins, and the efficiency of unmasking with pronase or trypsin, therefore, varied with the location in the tissue. The J chain was particularly vulnerable to degradation during proteolytic treatment. The extensive masking of extracellular immunoglobulin in formalin-fixed tissue afforded a relatively good signal-to-noise ratio for immunoglobulin-producing cells when kappa and lambda chains were traced. Thus, differentiation between polyclonal and monoclonal B-cell processes on the basis of cytoplasmic labelling was often better in undigested sections. However, the light-chain type of membrane immunoglobulin could usually not be determined in directly fixed tissue. Ethanol fixation preceded by washing in saline afforded such determination and also preserved certain T-cell and HLA-DR antigens as well as diffuse alpha-naphthylbutyrate esterase. Reactive and malignant macrophages could further be traced by their cytoplasmic expression of L1 antigen, both in formalin- and ethanol-fixed material.
对八种交联固定剂进行了测试，以评估其对细胞外或细胞内IgG、IgA、IgM、IgD、κ和λ轻链、J链及分泌成分的保存效果。大多数所选固定剂已用于近期关于淋巴增殖性疾病的免疫组织化学研究，包括常规福尔马林、戊二醛（1%）-福尔马林、贝克氏福尔马林-钙、福尔马林-升汞、乙酸（2%）-福尔马林-盐水、布安氏液、苏萨固定剂和碳二亚胺。通过免疫荧光法，将在含有定量IgG或IgA的人工测试底物以及生物底物（结肠黏膜、扁桃体和不同类型淋巴瘤）中获得的结果，与在冷96%乙醇中固定（有或无预固定48小时洗涤期）所提供的抗原保存情况进行比较。使用大多数其他固定剂进行检测时，所需抗原浓度至少要高八倍。布安氏液和苏萨固定剂的特殊之处在于，检测IgG时它们所需抗原浓度高150倍，但检测IgA时仅高3 - 8倍。除戊二醛外，所有固定剂对轻链的保存效果相对较好。对于所有交联固定剂，抗原掩盖程度取决于环境蛋白浓度，因此用链霉蛋白酶或胰蛋白酶进行去掩盖的效率会因组织位置而异。J链在蛋白水解处理过程中特别容易降解。在福尔马林固定的组织中，细胞外免疫球蛋白的广泛掩盖使得在追踪κ和λ链时，产生免疫球蛋白的细胞具有相对较好的信噪比。因此，基于细胞质标记区分多克隆和单克隆B细胞过程，在未消化切片中通常效果更好。然而，在直接固定的组织中，通常无法确定膜免疫球蛋白的轻链类型。先用盐水洗涤再进行乙醇固定可实现这种确定，同时还能保存某些T细胞和HLA - DR抗原以及弥漫性α-萘丁酸酯酶。在福尔马林和乙醇固定的材料中，反应性和恶性巨噬细胞均可通过其细胞质中L1抗原的表达进一步追踪。
