3 3 diaminobenzidine chromogen solution

Immunolocalization of AQP3, -7, -8 and -11 was evaluated in human sperm samples as previously described [63 (link)]. Samples were smeared on polylysine-coated slides, air dried and fixed in 4% paraformaldehyde in PBS for 30 min, washed with PBS and then treated with 0.3% hydrogen peroxide in methanol for 10 min at room temperature to block the endogenous peroxidases. After washing for 5 min with PBS, sections were blocked with 3% BSA in PBS for 30 min at room temperature. Slides were incubated for 3 h at room temperature with affinity pure primary antibodies (see Immunoblotting sections) diluted 1:200 in antibody diluent (Dako). After three 10 min washes with PBS containing 1% BSA, the sections were first incubated for 30 min at room temperature with biotinylated anti-rabbit IgG and then washed three times with PBS containing 1% BSA for 10 min at room temperature with HRP-conjugated streptavidin (Universal DAKO LSAB^® + kit, peroxidase, K0679, DakoCytomation, Milan, Italy). The reaction was visualized by incubation with a DakoCytomation 3,3′-diaminobenzidine chromogen solution. Negative controls were performed by incubating slices with non-immune serum.
The immunostained slides were examined by light microscopy using an Olympus BX41 and the digital images acquired with the Nikon DS-Fi1 digital camera using Nis Element F Imaging Software (2.33, Nikon, Tokyo, Japan).

Paraffin tissue sections were deparaffinized in two changes of xylene, rehydrated in graded ethanol, and treated for 30 min with 3% H₂O₂ solution in methanol to block endogenous peroxidase activity. Then, the sections were incubated with mouse monoclonal anti‐human TSPAN1 antibody (Santa Cruz Biotechnology, Inc., Dallas, TX, USA; Cat# sc‐376551) for 1 h at RT, followed by detection using Dako LSAB+ (Dako, Glostrup, Denmark). The reaction product was developed with 3,3′‐diaminobenzidine chromogen solution (Dako). Sections were counterstained with hematoxylin and mounted in Faramount aqueous mounting medium (Dako). We used human small intestine tissue as positive controls for TSPAN1 staining. TSPAN1 staining was confirmed at the cytoplasmic and apical membrane of glandular cells (Fig. S2). TSPAN1 staining was scored as positive when tumor or epithelial cells showed cytoplasmic and membrane immunoreactivity. It was performed by a gynecological pathologist. TSPAN1 staining results were scored based on intensity (0 = negative, 1 = weak, 2 = moderate, 3 = strong) and the percentage of positive cells (0 = 0%, 1 = 1–25%, 2 = 26–50%, 3 = 51–100%), as described previously [25].