Gill s hematoxylin 2

Human and NHP testis tissue samples were fixed in buffered formaldehyde or buffered Bouin's fixative and subsequently embedded in paraffin. Tissue sections, cut 4 or 5 µm thickness, were dewaxed and rehydrated in decreasing concentrations of ethanol.
Immunohistochemical staining was carried out with a standard indirect peroxidase method as described by Gueler et al (2012). Briefly, slides were pre-treated with 0.1 M boric acid pH 7 at 60°C overnight. After washing in permeabilization buffer (0.1 M Tris, 0.1 M NaCl, 0.1% Triton X-100; pH 7.4), endogenous peroxidase was quenched by incubation in 3% (v/v) hydrogen peroxide in methanol for 10 min at room temperature, followed by blocking with 3% (v/v) goat serum (DAKO, Glostrup, Denmark) in permeabilization buffer for 1 hour. All sections were incubated overnight at 4˚C with diluted primary DBY-10 antibodies (1:500 v/v) marking specifically DDX3Y proteins [7]. Subsequently, a secondary, biotinylated goat-antirabbit antibody (DAKO Cytomation, Glostrup, Denmark) was applied (1:100 v/v) followed by incubation with avidin-biotin complex (Vector Laboratories, Burlingame, CA, USA). Finally, slides were stained with DAB (3, 3'-diamino-benzidine tetrahydrochloride) counterstained with Gill's hematoxylin II (Roth, Karlsruhe, Germany) and mounted in Immuno-Mount (Shadon, Pittsburgh, PA, USA).