POSTFIXATION DETERGENT TREATMENT FOR IMMUNOFLUORESCENCE SUPPRESSES LOCALIZATION OF SOME INTEGRAL MEMBRANE-PROTEINS
POSTFIXATION DETERGENT TREATMENT FOR IMMUNOFLUORESCENCE SUPPRESSES LOCALIZATION OF SOME INTEGRAL MEMBRANE-PROTEINS
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DOI：
10.1177/33.8.3894499
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发表时间：
1985-01-01
影响因子：
3.2
通讯作者：
WILLINGHAM, MC
中科院分区：
文献类型：
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作者：
GOLDENTHAL, KL;HEDMAN, K;WILLINGHAM, MC
对培养的动物细胞进行免疫荧光显微镜观察时，通常在甲醛固定细胞膜后用去污剂透化处理，这样抗体才能接触到细胞内抗原。比较了几种去污剂在甲醛固定后影响细胞内蛋白质定位或使不同细胞器对抗体透化的能力。还使用了皂素，它是一种类似去污剂的分子，能使含胆固醇的膜透化。四种单克隆抗体仅用皂素处理时呈现明亮、离散的荧光定位，但当细胞用非离子型去污剂如Triton X - 100或NP - 40处理时几乎检测不到。这些IgG抗体包括2种针对溶酶体膜糖蛋白的抗体、1种针对质膜和内吞小泡中一种整合膜蛋白的抗体以及1种针对内质网和核膜中一种膜蛋白的抗体。定位于线粒体和细胞核的抗原需要使用如Triton X - 100这样的去污剂才能检测到。许多其他膜蛋白或胞质蛋白的检测不受Triton X - 100处理的影响。非离子型去污剂如Triton X - 100会导致一些膜蛋白检测出现假象性缺失。皂素是一种用于许多细胞器中细胞内膜抗原免疫荧光检测的良好替代试剂。
Immunofluorescence microscopy of cultured animal cells is often performed after detergent permeabilization of formaldehyde-fixed cellular membranes so that antibodies may have access to intracellular antigens. A comparison was made of the ability of several detergents, after formaldehyde fixation, to affect localization of intracellular proteins or to permeabilize different organelles to antibodies. Saponin, a detergent-like molecule that can permeabilize cholesterol-containing membranes, was also used. Four monoclonal antibodies had a bright, discrete fluorescence localization with saponin alone, but were almost undetectable when the cells were treated with nonionic detergents such as Triton X-100 or NP-40. These immunoglobulin G antibodies included 2 against lysosomal membrane glycoproteins, 1 against an integral membrane protein found in the plasma membrane and endocytic vesicles and 1 against a membrane protein in the endoplasmic reticulum and the nuclear envelope. Antigens localized in mitochondria and the nucleus required the use of a detergent such as Triton X-100 for their detection. The detection of a number of other membrane or cytoplasmic proteins was unaffected by Triton X-100 treatment. Nonionic detergents such as Triton X-100 cause artifactural loss of detection of some membrane proteins. Saponin is a favorable alternative reagent for immunofluorescence detection of intracellular membrane antigens in many organelles.