Nishida Haruto, Kashima Kenji, Yano Shinji, Daa Tsutomu, Arakane Motoki, Oyama Yuzo, Kusaba Takahiro, Kadowaki Hiroko, Yokoyama Shigeo
Department of Diagnostic Pathology, Faculty of Medicine, Oita University, Yufu, Japan.
Appl Immunohistochem Mol Morphol. 2019 May/Jun;27(5):e42-e47. doi: 10.1097/PAI.0000000000000735.
We herein introduce a novel method of biotin tagging immunoelectron microscopy for formalin-fixed, paraffin-embedded sections. This method was developed to utilize the antigenicity of biotin on epoxy-embedded ultrathin sections that could readily be recovered by a previously established antigen retrieval method as most monoclonal antibodies failed to recognize their targets by immunoelectron microscopy following antigen retrieval. The biotin tagging method was composed of preembedding immunostaining, epoxy-embedding and sectioning, and postembedding immunostaining steps. The preembedding step utilized the streptavidin-biotin-peroxidase complex method for immunohistochemistry to tag every antigen with a biotin in 3-μm thick paraffin-embedded sections. Next, fixation and processing for transmission electron microscopy (TEM) were performed on sections on glass slides, and ultrathin sections were prepared in epoxy-embedded blocks. In the postembedding step, antigen retrieval was followed by serial incubations with an antibiotin monoclonal antibody and anti-mouse IgG-labeled gold particles. The results obtained using antibodies against a variety of intracellular targets were satisfactory; positive gold particles were observed corresponding to targeted intracellular structures. This study demonstrated that the biotin tagging method was a convenient approach for successful labeling of paraffin-embedded sections for TEM using monoclonal antibodies, although it has relatively poor subcellular labeling quality.
我们在此介绍一种用于福尔马林固定、石蜡包埋切片的生物素标记免疫电子显微镜新方法。开发此方法是为了利用环氧包埋超薄切片上生物素的抗原性，由于大多数单克隆抗体在抗原修复后通过免疫电子显微镜无法识别其靶标，而通过先前建立的抗原修复方法可以很容易地恢复这种抗原性。生物素标记方法由包埋前免疫染色、环氧包埋和切片以及包埋后免疫染色步骤组成。包埋前步骤利用链霉亲和素-生物素-过氧化物酶复合物免疫组织化学方法，在3μm厚的石蜡包埋切片中用生物素标记每个抗原。接下来，对载玻片上的切片进行透射电子显微镜（TEM）固定和处理，并在环氧包埋块中制备超薄切片。在包埋后步骤中，抗原修复后依次用抗生物素单克隆抗体和抗小鼠IgG标记的金颗粒孵育。使用针对多种细胞内靶标的抗体获得的结果令人满意；观察到与靶向细胞内结构相对应的阳性金颗粒。本研究表明，生物素标记方法是一种使用单克隆抗体成功标记石蜡包埋切片用于TEM的便捷方法，尽管其亚细胞标记质量相对较差。
