Sato Y, Ito T, Nozawa A, Kanisawa M
Department of Pathology, Yokohama City University School of Medicine, Japan.
Biotech Histochem. 1995 Jul;70(4):169-74. doi: 10.3109/10520299509107308.
To establish bromodeoxyuridine (BrdUrd)/iododeoxyuridine (IdUrd) double immunostaining for thick sections of epoxy resin-embedded tissues, young hamsters received intra-peritoneal injections of IdUrd and BrdUrd 3 hr and 1 hr before sacrifice, respectively. The intestines were fixed with phosphate-buffered 4% paraformaldehyde and embedded in an Epon-Araldite mixture. The epoxy resin was removed completely by a sodium methoxide/benzene/methanol solution. This epoxy resin removal method was effective for BrdUrd/IdUrd immunostaining using a mono-specific antibody for BrdUrd (Br-3) and a bi-specific antibody for BrdUrd (Br-3) and a bi-specific antibody for BrdUrd and IdUrd (IU-4), followed by the ABC complex method. Epoxy sections stained with these antibodies showed clear localization of nuclei incorporating the two thymidine analogues with precise morphology of labeled cells. Furthermore, ultrastructural observation of thin sections adjacent to thick sections immunostained for BrdUrd/IdUrd confirmed the cell type and ultrastructural features of cells labeled with these thymidine analogues.
为建立用于环氧树脂包埋组织厚切片的溴脱氧尿苷（BrdUrd）/碘脱氧尿苷（IdUrd）双重免疫染色方法，幼年仓鼠在处死前3小时和1小时分别腹腔注射IdUrd和BrdUrd。肠道用磷酸盐缓冲的4%多聚甲醛固定，然后包埋于环氧树脂-阿拉地特混合物中。用甲醇钠/苯/甲醇溶液完全去除环氧树脂。这种去除环氧树脂的方法对于使用针对BrdUrd的单特异性抗体（Br-3）以及针对BrdUrd和IdUrd的双特异性抗体（IU-4）进行BrdUrd/IdUrd免疫染色有效，随后采用ABC复合物法。用这些抗体染色的环氧树脂切片显示出掺入两种胸苷类似物的细胞核的清晰定位以及标记细胞的精确形态。此外，对与经BrdUrd/IdUrd免疫染色的厚切片相邻的薄切片进行超微结构观察，证实了用这些胸苷类似物标记的细胞的细胞类型和超微结构特征。
