Dako 3 3 diaminobenzidine substrate

Slides having 4 μm thick testis sections attached to them were immersed in a pressure cooker filled with tris-EDTA buffer and 0.05% tween 20 (pH 9.0), to retrieve antigens for 3 min. Thereafter, endogenous peroxidase was blocked using 3% hydrogen peroxide in phosphate buffered saline for 5 min, followed by washing with distilled water and tris-buffered saline containing 0.05% tween 20 (TBST, pH 8.4). Further, slide containing testis sections were incubated with polyclonal primary antibodies for PCNA (1:50), cleaved caspase-3 (1:150), NF-κB (p65) (1:80), IL-10 (1:40), IL-1β (1:80), and TNF-α (1:120) (Cloud-Clone Corp, USA) for 24 h at 4 °C. Testis sections were incubated with Dako EnVision™+ System/HRP labelled polymer containing goat anti-rabbit secondary antibody (Agilent Technologies, Inc. United States of America) for 30 min at room temperature after washing with TBST. With the aid of Dako 3,3′-diaminobenzidine substrate (Agilent Technologies, Inc. USA) visualisation was conducted for 5 min at room temperature. Thereafter, hematoxylin was employed to counter stain the sections for 5 s. They were dehydrated and viewed using a light microscope (Olympus BX41, United Kingdom). ImageJ software (ImageJ, NIH—Bethesda, MD, USA) was used to analyse the photographs for area and intensity of brown stain, and also to evaluate the quantitative data of the stained area.

Four-micrometer-thick sections of testis samples were placed into a pressure cooker containing Tris–EDTA buffer with 0.05% tween 20 (pH 9.0), for 3 min for antigen retrieval, followed by blocking of endogenous peroxidase with 3% hydrogen peroxide in phosphate-buffered saline for 5 min, then washing with distilled water and Tris-buffered saline containing 0.05% tween 20 (TBST, pH 8.4). Thereafter, sections were incubated with polyclonal primary antibodies for TNF-α (1:120)  Cat. No. A356015, caspase-3 (1:150) Cat. No. PK-CA577-K16, and PCNA (1:50) Cat. No. OKCD02760 (Cloud-Clone Corp, USA) for 24 h at 4 °C. After washing with TBST, sections were incubated with Dako EnVision™ + System/HRP-labeled polymer containing goat anti-rabbit secondary antibody (Agilent Technologies, Inc. USA) for 30 min at room temperature. Visualization was performed using Dako 3,3′-diaminobenzidine substrate (Agilent Technologies, Inc. USA) for 5 min at room temperature. Sections were counter-stained in hematoxylin for 5 s, dehydrated, and viewed using a light microscope (Olympus BX41, UK). Quantitative measurement of the percentage area of TNF-, as well as caspase-3 and PCNA immunostaining color intensity, was done by analyzing the intensity of the brown stain in the image using ImageJ software. (ImageJ, NIH-Bethesda, MD, USA).

Antigen retrieval was performed on 4 µm thick sections using a pressure cooker containing Tris-EDTA buffer with 0.05% Tween 20 (pH 9.0) for 3 min. Thereafter, endogenous peroxidase was blocked by incubating the sections with 3% hydrogen peroxide in PBS for 5 min, followed by washing with distilled water and Tris-buffered saline containing 0.05% Tween 20 (TBST, pH 8.4). Incubation with polyclonal primary antibodies for NF-κB (1:80), TNF-α (1:120), IL-1β (1:80), IL-10 (1:40) and caspase-3 (1:150) was done at 4 °C overnight. After washing twice with TBST, sections were incubated with Dako EnVision™+ System/HRP labeled polymer containing goat anti-rabbit secondary antibody (Agilent Technologies, Inc., Santa Clara, CA, USA) for 30 min at room temperature. Visualization was performed using Dako 3,3′-diaminobenzidine substrate (Agilent Technologies, Inc., Santa Clara, CA, USA) at room temperature for 5 min. Thereafter, the sections were counterstained for 5 sec with hematoxylin, dehydrated and examined under a light microscope (Olympus BX41, Tokyo, Japan). The brown staining intensity was determined using ImageJ software (ImageJ, NIH-Bethesda, MD, USA).