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

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.

Testicular sections of 5 µm were used for caspase-3 and PCNA immunostaining. Tris–EDTA buffer with 0.05% tween 20, pH 9.0 was used for antigen retrieval in a pressure cooker for 5 min. Thereafter, endogenous peroxidase blocking was performed using 3% H₂O₂ in PBS for 5 min. Sections were incubated overnight at 4 °C with rabbit polyclonal primary antibodies for caspase-3 (PAA626Ra01, 1:150) and PCNA (PAA591Mi01, 1:50) (Cloud-Clone Corp, Houston, TX, USA) after rinsing with distilled water and tris-buffered saline containing 0.05% tween 20 (TBST, pH 8.4). Sections were rinsed twice for 5 min with TBST and incubated at room temperature for 30 min using Dako EnVision + System/HRP-labelled polymer containing goat anti-rabbit secondary antibody (catalogue number: K4003, Agilent Technologies, Inc. Santa Clara, USA). DAB substrate (Agilent Technologies, Inc. Santa Clara, USA) was then utilised for detection. Sections were counter-stained with haematoxylin and viewed under a light microscope (Olympus BX41, Olympus Corporation, Tokyo, Japan). ImageJ software (NIH-Bethesda, MD, USA) was used to analyse the intensity of staining and percentage positive cells, for both caspase-3 and PCNA proteins. The results were expressed as fold change relative to the normal control group.