Dual immunofluorescence labeling with cell-specific markers localizes BRCA1 in both basal and luminal epithelial cells in primary outgrowth from noncancerous mammary ductal and alveolar preparations.
Dual immunofluorescence labeling with cell-specific markers localizes BRCA1 in both basal and luminal epithelial cells in primary outgrowth from noncancerous mammary ductal and alveolar preparations.
复制标题
使用细胞特异性标记物的双重免疫荧光标记将 BRCA1 定位于非癌性乳腺导管和肺泡制剂的初级生长物中的基底和管腔上皮细胞中。
DOI：
10.1007/s004419900067
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发表时间：
1999
影响因子：
3.6
通讯作者：
Daniel,DC
中科院分区：
文献类型：
--
作者：
Daniel,DC
两种分化的乳腺上皮细胞类型在人类乳腺癌进展中所起的作用目前尚未确定。这项工作解决了乳腺肿瘤抑制基因产物BRCA 1是否定位于从乳腺类器官培养的非癌产物中的基底和/或腔上皮细胞的问题。直接使用导管和肺泡制备物的原代上皮细胞生长物，以便于在接近完整组织的条件下进行小规模分析。共聚焦显微镜下，BRCA 1免疫荧光检测到的上皮生长的细胞核中的大部分。标记强度高的细胞核呈点状染色。用小鼠IgG作为阴性一抗对照或用抗细胞膜受体ErbB 2的一抗仅观察到最小的非特异性染色，据报道ErbB 2在乳腺癌中表达，但在正常乳腺组织中不可检测或弱表达。双重标记用于区分BRCA 1染色的上皮细胞类型。用抗波形蛋白单克隆抗体鉴定基底细胞，用抗细胞角蛋白19单克隆抗体鉴定管腔细胞。使用抗BRCA 1的单克隆抗体与这些标记物中的每一种进行共标记。荧光显微镜显示BRCA 1免疫反应在基底和管腔间期细胞。BRCA 1免疫荧光在有丝分裂期弥漫分布于染色体团周围。
The role played by either of the two differentiated mammary epithelial cell types in human breast cancer progression is currently not defined. This work addresses the question of whether the mammary tumor suppressor gene product BRCA1 is localized in basal and/or luminal epithelial cells in noncancerous outgrowth cultured from breast organoids. Primary epithelial cell outgrowths from ductal and alveolar preparations were directly employed to facilitate small-scale analysis under conditions closely approximating intact tissue. BRCA1 immunofluorescence was detected for the most part in cell nuclei of the epithelial outgrowth when using confocal microscopy. Nuclear staining was punctate in the cells with higher labeling intensity. Only minimal nonspecific staining was observed with mouse IgG as a negative primary antibody control or with primary antibody against the cell membrane receptor ErbB2, reported to be expressed in breast cancer, but was either not detectable or weakly expressed in normal breast tissue. Dual labeling was used to distinguish which epithelial cell type(s) stains for BRCA1. Primary monoclonal antibody against vimentin was used to identify basal cells, while antibody against cytokeratin 19 was used to identify luminal cells. Monoclonal antibody against BRCA1 was used for colabeling with each of these markers. Epifluorescence microscopy revealed BRCA1 immunoreactivity in both basal and luminal interphase cells. BRCA1 immunofluorescence was diffusely located about the chromosome mass during mitosis.