Serial sections of 3-μm thick paraffin-embedded testis were prepared and mounted on poly-L-lysine slides. The sections were deparaffinized in xylene and then rehydrated in graded alcohols.
The tissue sections were heated in a microwave oven in pre-heated 10 mM sodium citrate buffer pH 6.0 for 5 min for antigen retrieval and permeabilized with 0.1% Triton-X 100 in PBS for 30 min. The slides were washed three times with PBS before incubation in Blocking One Solution (Nacalai Tesque, Kyoto, Japan) for 10 min to block non-specific binding. The slides were rewashed with PBS three times. The tissue sections were incubated overnight at 4°C with mouse monoclonal phosphohistone 3 (pHH3) antibody conjugated with Alexa Flour 594 (Santa Cruz Biotechnology, Texas, United States) at a dilution of 1:100.
After antibody binding, the slides were washed three times with PBS. DNA was counterstained with 10 μg/mL Hoechst 33342 and observed using Nikon Eclipse Ni fluorescent microscope under 400 × magnification. About 10–15 random fields were captured using Nikon Y-T TV. The intensity of the staining of 20 random seminiferous tubules was measured using Image J software v1.52a. The steps were repeated for rectum adenocarcinoma tissue (Kim et al., 2018 (link)) as a positive control for the staining.
The tissue sections were heated in a microwave oven in pre-heated 10 mM sodium citrate buffer pH 6.0 for 5 min for antigen retrieval and permeabilized with 0.1% Triton-X 100 in PBS for 30 min. The slides were washed three times with PBS before incubation in Blocking One Solution (Nacalai Tesque, Kyoto, Japan) for 10 min to block non-specific binding. The slides were rewashed with PBS three times. The tissue sections were incubated overnight at 4°C with mouse monoclonal phosphohistone 3 (pHH3) antibody conjugated with Alexa Flour 594 (Santa Cruz Biotechnology, Texas, United States) at a dilution of 1:100.
After antibody binding, the slides were washed three times with PBS. DNA was counterstained with 10 μg/mL Hoechst 33342 and observed using Nikon Eclipse Ni fluorescent microscope under 400 × magnification. About 10–15 random fields were captured using Nikon Y-T TV. The intensity of the staining of 20 random seminiferous tubules was measured using Image J software v1.52a. The steps were repeated for rectum adenocarcinoma tissue (Kim et al., 2018 (link)) as a positive control for the staining.
Free full text: Click here
