Jilin Province Key Laboratory of Animal Embryo Engineering, College of Animal Sciences and Veterinary Medicine, Jilin University, Changchun 130062, China.
Microsc Res Tech. 2012 Aug;75(8):1150-3. doi: 10.1002/jemt.22080. Epub 2012 Jun 18.
Whole-mount immunohistochemistry (whole-mount IH) of the seminiferous tubule is widely used to investigate the self-renewal and differentiation of spermatogonial stem cells (SSCs). Examination of the length of spermatogonial cysts is critical for tracing SSCs lineage by using Whole-mount IH. However, it is difficult for antibody molecules to penetrate into the depth of seminiferous epithelium because its thickness and the tight peritubular myoid and basement membrane outside. Here, we developed a free-floating immunofluorescent procedure of mouse seminiferous tubules using regular incubation time and normal antibody concentration. Microscopic results showed that undifferentiated spermatogonia were positively labeled by promyelocytic leukemia zinc finger protein, E-cadherin, and glial cell line-derived neurotrophic factor family receptor alpha 1, respectively. Spermatogonial cysts in varied length were revealed clearly and spermatogonia subpopulations including A(single) (A(s)), A(paired) (A(pr)), and A(aligned) (A(al)) were distinguished in lower background images. This method provides us an alternate simple way to trace the lineage of individual SSCs and show their three-dimensional locations and distributions within their niches anatomically in next step.
全组织免疫荧光化学（whole-mount IH）技术广泛应用于研究精原干细胞（SSCs）的自我更新和分化。通过全组织免疫荧光化学技术追踪 SSCs 谱系时，对精原细胞小囊长度的检测至关重要。然而，由于生精上皮的厚度以及管周肌样细胞和外部基膜的紧密性，抗体分子很难穿透到生精上皮的深度。在这里，我们开发了一种使用常规孵育时间和正常抗体浓度的游离免疫荧光组织学方法。显微镜结果表明，原始细胞白血病锌指蛋白、E-钙黏蛋白和神经胶质细胞系源性神经营养因子家族受体α 1分别对未分化的精原细胞进行阳性标记。清晰地显示了不同长度的精原细胞小囊，并在低背景图像中区分了精原细胞亚群，包括 A（单个）（A(s)）、A（配对）（A(pr)）和 A（对齐）（A(al)）。该方法为我们提供了一种替代的简单方法，可以追踪单个 SSCs 的谱系，并在下一步中在其解剖位中显示它们在其龛位中的三维位置和分布。
