Bs 1329r

Immunohistochemical staining was conducted based on the procedures in our previous report [4 (link)]. Specifically, the overnight incubation (4 °C) of testicular tissue sections was executed by virtue of the rabbit polyclonal antibody against ZO-1 (diluted at 1:100; bs-1329R; Bioss) or LC3 (1:100 dilution; 14600-1-AP, Proteintech) in combination with the mouse monoclonal SOX9 antibody (diluted at 1:100; ab76997; abcam, an SC-specific marker). Later, the tissue sections, washed 3 times in PBS, were cultured for 45 min using the FITC-coupled goat anti-rabbit IgG (H+L) antibody (1:200 dilution; HS111, TransGen, Beijing, China) and the PE-labeled goat anti-mouse IgG (H+L) antibody (1:200 dilution; HS221, TransGen, Beijing, China). Lastly, the cell nuclei received DAPI staining based on the previously described methods, followed by observation and photography of the sections under the Olympus-DP73 optical microscope (Tokyo, Japan).

First, the anti-SOX9 mouse monoclonal antibody (a specific SC marker) together with the rabbit polyclonal antibody against ZO-1 (diluted at 1 : 100; bs-1329R; Bioss), Claudin-11 (dilution rate 1 : 100; bs-21509R; Bioss), or Occludin (diluted at 1 : 100; bs-10011R; Bioss) was applied to incubate the testicular tissue sections (4 μm) at 4°C overnight. Subsequently, the tissue sections were cleaned by PBS for 3 times, and the goat anti-rabbit IgG (H + L) antibody conjugated with FITC (diluted at 1 : 200; TransGen, Beijing, China) and the goat anti-mouse IgG (H + L) antibody labeled with PE (dilution rate 1 : 200; TransGen, Beijing, China) were utilized for 45 min of incubation. Finally, Hoechst 33342 was adopted to stain the cell nuclei as per the methods described before [28 (link)]. A fluorescence microscope (Nikon, Tokyo, Japan) was employed to observe the protein expression and colocalization.