Periodic acid schiff staining kit

Goblet cells were stained using the periodic acid Schiff staining kit (Beyotime, Shanghai, China) [42 (link)]. Briefly, the paraffin sections of each group were first dewaxed with xylene and gradient ethanol, cleaned with distilled water, purified with iodate for 10 min, rinsed with tap water for 10 min and Schiff solution for 10 min, rinsed with PBS for 5 min, and nucleated with harisoxylin or Meyer hematoxylin for 3 min (too deep nuclear staining can be differentiated by hydrochloric acid alcohol). They were rinsed with running water for 5 min. Finally, routine dehydrated by gradient ethanol and xylene, sealed with neutral resin, microscopic observation, and photography records. A total of 30 experimental animals C57BL/6 mice were used.

After fixation, all the ovarian tissues were deparaffinized and rehydrated through a series of solutions from 100 to 70% ethanol and deionized water. To perform follicle counting, individual left ovarian tissues were serially sectioned parasagittally at 6 μm and stained with a periodic acid-Schiff staining kit (Beyotime Biotechnology Co., Ltd, Beijing, China) based on the finding that all modern crocodilians have two functional ovaries in gametogenesis and that the number of germ cells did not differ between the two ovaries [16 (link)]. To quantify the exact number of germ cells belonging to different developmental stages, the number of oogonia/oocytes within the Nest and Pmf was counted via complete serial section examination under the microscope, and the numbers of oocytes within the PF (19 to 22 μm in diameter) and Pre-VF (52 μm in diameter) were counted every three sections (18 μm) and nine sections (54 μm), respectively. Using this method as a reference, the number of germ cells belonging to different meiosis stages was also determined to calculate the dynamic changes in germ cells within different meiosis processes.