Paraffin slicing machine

Tumors were excised, fixed in 4% paraformaldehyde (PFA), dehydrated with a graded ethanol series, cleared in dimethylbenzene, and embedded in paraffin. Next, tissue blocks were cut into 4-μm sections by using a paraffin-slicing machine (Leica, Nussloch, Germany), and mounted on glass slides. Tissue sections were deparaffinized and rehydrated, heated in citrate buffer (0.01 M, pH 6.0), and treated with endogenous peroxidase at room temperature. After blocking in 10% goat serum, the sections were stained with rabbit polyclonal anti-Cyclin B₁ (1:200) or mouse monoclonal anti-p53 (1:400) primary antibodies and incubated overnight at 4 °C. Subsequently, sections were incubated with secondary antibodies and stained with DAB reagent. Finally, all sections were observed under an electron microscope. Immunopositive expression in cells was quantified with IOD values by using IPP 6.0.

A paraffin slicing machine (Leica, Germany) was used to cut paraffin-embedded samples into 4 μm slices. IHC staining was performed following the standard protocol as previously described [18 (link)]. After initial deparaffinization, antigen retrieval, and blockade, the anti-UBE2T antibody (cat#: ab154022, Abcam, Cambridge, MA, United States) was applied to sections overnight at 4 °C in a humidified container. A horseradish peroxidase-labeled secondary antibody (cat#: ab205718, Abcam, Cambridge, MA, United States) followed the next day. DAB (50:1, Novus Biologicals, Centennial, CO, United States) was used to visualize UBE2T staining, followed by hematoxylin counterstain. Sections skipping the primary antibody were used as the negative control. Each sample was designated a score of 0 (negative), 1 (mild), 2 (moderate), or 3 (strong) based on the relative staining intensity. Tissue with score ≤ 1 or ≥ 2 was defined as low and high expression, respectively.

After fixed in Bouin’s solution for 24 h, the testis was orderly dehydrated using gradient alcohol. Then, the testis was embedded by paraffin until transparent, then dealt with xylene. The paraffin blocks were cut into slices with 5 μm using a paraffin slicing machine (Lecia, Wetzlar, Germany). Finally, the nucleus and cytoplasm were stained by hematoxylin and eosin, respectively, and the sections were observed by light microscopy (Nikon IQ50, Tokyo, Japan) to classify the gonadal stages.

We used a paraffin slicing machine (Leica, Germany) to cut paraffin slices at about 4 microns. The slices were baked in a 67°C oven for 2 h and deparaffinized in xylene and rehydrated in graded ethanol, then boiled in citrate buffer (pH 6.0) for 3 min at 100°C and cooled naturally to room temperature. We immersed the sections in 3% H₂O₂ for 10 min to block the endogenous peroxidase and washed with PBS. Sections were further incubated with anti-UBE2T antibody (cat#: ab154022, Abcam, Cambridge, MA, United States) overnight at 4°C in a humidified container. The next day, the sections were washed with PBS three times and then incubated with a horseradish peroxidase-labeled secondary antibody (cat#: ab205718, Abcam, Cambridge, MA, United States) for 1 h at room temperature. Sections were washed with PBS three times again. A drop of DAB (50:1, Novus Biologicals, Centennial, CO, United States) was added to every section, which was observed under microscopy for timely termination. Hematoxylin was used to counterstain sections briefly, which were observed under a microscope. Finally, sections were dehydrated in ethanol and sealed with neutral resin. In the case of negative control, the primary antibody was omitted. The score of relative staining intensity was 0, 1, 2, 3, and 4. Tissue with score ≤1 or ≥2 was defined as low and high expression, respectively.