背景:睾丸组织固定的方式直接影响结缔组织和生精小管之间的相关性和结构完整性,这对研究男性生殖发育至关重要。本研究旨在寻找最佳的固定剂和固定时间,以产生高质量的睾丸组织病理学切片,为利用数字病理技术深入研究男性生殖发育提供了合适的基础。
方法:从25只雄性C57BL/6小鼠的两侧取出睾丸。将样品固定在三种不同的固定剂中,10%中性缓冲福尔马林(10%NBF),改性戴维森流体(mDF),和布恩流体(BF),8、12和24小时,分别。苏木精和伊红(H&E)染色,高碘酸希夫-苏木精(PAS-h)染色,和免疫组织化学(IHC)用于评估睾丸形态,小鼠生精小管分期,和蛋白质保存。AperioScanScopeCS2全景扫描用于进行定量分析。
结果:H&E染色显示10%NBF导致生精上皮厚度减少约15-17%。当用PAS-h染色顶体时,BF和mDF提供优异的结果。与BF固定的样品相比,mDF中突触复合体3(Sycp3)的IHC染色更好。与10%NBF相比,mDF和BF中的固定改善了睾丸组织形态。
结论:定量分析显示BF表现出非常低的IHC染色效率,并显示小鼠睾丸用mDF固定12小时,表现出形态学细节,PAS-h染色对生精小管分期的优异效率,和IHC结果。此外,随着固定时间的延长,睾丸的形态损伤延长。
BACKGROUND: The way of testicular tissue fixation directly affects the correlation and structural integrity between connective tissue and seminiferous tubules, which is essential for the study of male reproductive development. This study aimed to find the optimal fixative and fixation time to produce high-quality testicular histopathological sections, and provided a suitable foundation for in-depth study of male reproductive development with digital pathology technology.
METHODS: Testes were removed from both sides of 25 male C57BL/6 mice. Samples were fixed in three different fixatives, 10% neutral buffered formalin (10% NBF), modified Davidson\'s fluid (mDF), and Bouin\'s Fluid (BF), for 8, 12, and 24 h, respectively. Hematoxylin and eosin (H&E) staining, periodic acid Schiff-hematoxylin (PAS-h) staining, and immunohistochemistry (IHC) were used to evaluate the testicle morphology, staging of mouse seminiferous tubules, and protein preservation. Aperio ScanScope CS2 panoramic scanning was used to perform quantitative analyses.
RESULTS: H&E staining showed 10% NBF resulted in an approximately 15-17% reduction in the thickness of seminiferous epithelium. BF and mDF provided excellent results when staining acrosomes with PAS-h. IHC staining of synaptonemal complexes 3 (Sycp3) was superior in mDF compared to BF-fixed samples. Fixation in mDF and BF improved testis tissue morphology compared to 10% NBF.
CONCLUSIONS: Quantitative analysis showed that BF exhibited a very low IHC staining efficiency and revealed that mouse testes fixed for 12 h with mDF, exhibited morphological details, excellent efficiency of PAS-h staining for seminiferous tubule staging, and IHC results. In addition, the morphological damage of testis was prolonged with the duration of fixation time.