Stahler M S, Pansky B, Budd G C
Biol Reprod. 1986 Nov;35(4):1075-80. doi: 10.1095/biolreprod35.4.1075.
Insulin-like immunoreactivity was localized in tissue sections and cell cultures of mouse seminal vesicle using the indirect technique of immunocytochemistry. Seminal vesicles were cut into fragments, fixed in 2.5% glutaraldehyde, embedded in epoxy resin, sectioned at 1 micron, and transferred to glass slides. Epithelial cell cultures of seminal vesicle were grown on coverslips in Dulbecco's Minimal Essential Medium for 4-6 days and fixed in 2.5% glutaraldehyde. Sections (etched with sodium ethanolate) or coverslips were incubated in guinea pig antiporcine insulin antiserum, in antiserum immunoabsorbed with porcine insulin, or in normal guinea pig serum. For indirect immunocytochemistry, incubation with primary antiserum was followed by treatment with rabbit anti-guinea pig immunoglobulin (Ig) G conjugated to peroxidase, or with protein A and then rabbit peroxidase anti-peroxidase (PAP). Finally, treated samples were incubated in phenylenediamine-pyrocatechol-H2O2 substrate mixture for 6-8 min at room temperature. Specific immunoreactivity to insulin antisera was confined to the epithelium of the seminal vesicle in tissue sections. No staining occurred in subepithelial connective tissue. Specific immunoreactivity was also observed in the cytoplasm of cultured seminal vesicle epithelial cells.
采用免疫细胞化学间接技术，将胰岛素样免疫反应定位在小鼠精囊的组织切片和细胞培养物中。将精囊切成碎片，固定于2.5%戊二醛中，包埋于环氧树脂中，切成1微米厚的切片，转移至载玻片上。精囊上皮细胞培养物在盖玻片上于杜氏改良伊格尔培养基中培养4 - 6天，然后固定于2.5%戊二醛中。切片（用乙醇钠蚀刻）或盖玻片分别用豚鼠抗猪胰岛素抗血清、用猪胰岛素免疫吸附的抗血清或正常豚鼠血清孵育。对于间接免疫细胞化学，在用一抗孵育后，接着用与过氧化物酶偶联的兔抗豚鼠免疫球蛋白（Ig）G处理，或用蛋白A处理，然后用兔过氧化物酶抗过氧化物酶（PAP）处理。最后，将处理过的样品在苯二胺 - 邻苯二酚 - H₂O₂底物混合物中于室温下孵育6 - 8分钟。对胰岛素抗血清的特异性免疫反应局限于组织切片中精囊的上皮。上皮下结缔组织未出现染色。在培养的精囊上皮细胞的细胞质中也观察到了特异性免疫反应。
