Dako envision system hrp labeled polymer containing goat anti rabbit secondary antibody

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).