Rm2245 slicer

Fully enclosed automatic dehydration machine (LEICA, Germany), automatic paraffin embedding machine (Germany, LEICA), ZMN-6802 pathological tissue bleaching and drying instrument (Changzhou Huali Electronic Co., Ltd.), RM2245 slicer (Germany, LEICA), BCD-509WD refrigerator (Haier, China), ophthalmic scissors, 18 cm U-shaped stainless steel pressure cooker (China Shunfa), one 1000-1500 w electric oven, high temperature-resistant plastic slicing rack 2 (Fuzhou Maixin), medium-sized, high-quality wolf-hair brush, ophthalmology curved regulator, etc., are the main instruments used in the study.

Testis samples for immunofluorescence staining were fixed in 4 % paraformaldehyde for 24 hours, then were dehydrated with graded alcohol series and embedded in paraffin. Cross-section of the testis was obtained using RM2245 slicer (LEICA, Germany). To observe the basic morphology of cross-section of the testis, hematocylin-eosin staining was performed according to the methods described in Rosenzweig [44 (link)]. The section was deparaffinized, rehydrated, and washed in phosphate-buffered solution (PBS) three times for immunofluorescence staining. Subsequently, the section was blocked with two drops of 3% H₂O₂ methanol solution for 10 min at room temperature, then washed in phosphate-buffered solution (PBS) for three times, and blocked with 100 μL of 5 % Bovine Serum Albumin (BSA) for 30 min at room temperature. After finishing the block, the section was incubated with MaltHSP70-2 antibody (diluted with PBS at a ratio of 1: 200 ) at 37 °C for 2 hours. Then the section was washed in PBS three times and incubated with TRITC-conjugated anti-rabbit IgG antibody (Beyotime, China) (diluted with TBST at a ratio of 1: 1000) at 37 °C for 1 hour. Finally, the section was stained using Diamidino-2-phenylindole (DAPI) and imaged on a confocal scanning fluorescence microscope DM2500 (LEICA, Germany). The excitation wavelength for MaltHSP70-2 was 549 nm, and for DAPI was 450 nm.