Avidin biotin hrp complex

Tumor tissues obtained from in vivo studies were rinsed in PBS and fixed in 4% paraformaldehyde. Samples were dehydrated in gradient ethanol, paraffin embedded, and sectioned (4 μm). Deparaffinized sections were stained with primary antibodies. The samples were incubated overnight with biotinylated secondary antibodies. Detection was done with avidin-biotin-HRP complex (Thermo scientific, Fremont, CA) and DAB as chromogen. Nuclei were counterstained with hematoxylin. The positive cells were counted in six fields per tumor sample. Results are expressed as the average ± S.D. of tumors per group. For TUNEL assay, sections were permeabilized with 0.1% Trition X-100 plus 0.1% sodium citrate and then incubated with 50 ml TUNEL reaction mixture (Roche) at 37 °C for 60 min. After rinsing with PBS three times, 50 ml converter-POD was added and the tissue cells were incubated in a humidified chamber for 30 min at 37 °C. DAB substrate was then added, followed by counterstaining with hematoxylin. The assay included negative controls (without terminal transferase). Apoptosis was quantified by counting the number of TUNEL-positive cells in at least six non-overlapping high-power fields on each section and evaluated.

Serial sections of vaginal tissue were deparaffinized and rehydrated. Sections were subjected to Heat Induced Epitope Retrieval in 10 mM Sodium Citrate buffer, 0.05% Tween-20, pH 6.0. The sections were heated and maintained at a temperature above 100° C for 10 mins, cooled, and permeabilized with 0.1% Triton-X-100 for 10 mins. The sections were washed and blocked with 10% normal goat serum. Rabbit anti-HSV-1 (Abcam) was used to detect HSV-1 antigen in the tissue. HSV-antibody negative, isotype-control IgG served as a control. Antibodies were incubated overnight at 4°C, washed, and goat anti-rabbit biotin secondary antibody added for 60 min at room temperature followed by avidin-biotin-HRP complex (Thermo Fisher Scientific) for 30 min. 3,3′-Diaminobenzidine (Thermo Fisher Scientific) was used to detect the HSV-1 antigen, counter-strained with hematoxylin, dehydrated, cleared, and mounted with cytoseal 60 (Electron Microscopy Sciences). Slides were imaged with a 10X objective. To measure the number of HSV-1 antigen foci, slides were imaged with a 2X objective, imported into Image J and manually counted.