Ultra sensitive abc peroxidase mouse igg staining kit

As previously described, tissue-embedded paraffin blocks were sectioned on positively charged slides, deparaffinized, and rehydrated. Citrate buffer (pH 6.0) was used for heat-induced antigen retrieval. Endogenous peroxidase was blocked with H₂O₂ and endogenous biotin with the aid of a blocking buffer (Cat no 32052, Thermofisher Scientific, USA). The sections were treated with primary antibodies against Caspase-3 (Cat no 9662, Cell Signaling Technology, MA, USA) with a dilution of (1:1000), and CD34 (Cat no 3569, Cell Signaling Technology, MA, USA) with a dilution of (1:1000). Immunoreactions were developed using the Ultra-sensitive ABC Peroxidase Mouse IgG Staining Kit (Cat no 32052, Thermofisher Scientific, USA) using the biotinylated secondary antibody. DAB (Cat no 34065, Thermofisher Scientific, USA) was used as chromogen, and the tissue was counterstained with Mayer's Hematoxylin.
Caspase-3 stained sections were assessed by counting the number of positively stained cells (cytoplasmic) in 10 HPFs at the invasive front of the tumor, away from the central areas of necrosis. CD34 stained sections were assessed by counting the number of positively stained endothelial cells lining vascular spaces within the tumor in 10HPFs. All assessments were conducted using the Leica Application Suite, Version 4.12.0 (Leica Microsystems CMS GmbH) image analysis software.

Endogenous peroxidases were quenched (3% hydrogen peroxide in methanol) followed by blocking (4% normal horse serum) for one hour in a humidified chamber. Sections were then incubated with mouse monoclonal anti-nitrotyrosine antibody (1:500, ab61392, Abcam) overnight at 4 °C. The Ultra-Sensitive ABC Peroxidase Mouse IgG Staining Kit (Thermo Fisher Scientific) was used for the application of the secondary antibody as well as the ABC complex. Sections were developed with 3–3′-diaminobenzidine (Sigma-Aldrich) and counterstained with hematoxylin for imaging using light microscopy. Images were captured using a Nikon Eclipse e400 microscope outfitted with a Nikon Coolpix 990 digital camera. Using the 40 × /0.65 N.A. air objective lens, sequential images of the tissue sections were taken to capture the entire length and depth of the articular cartilage across both the medial and lateral femoral condyles/tibial plateaus.

Neutral buffered formalin-fixed, paraffin-embedded brain tissue slices (5 μm thick) were used for immunohistochemical staining. The tissue slides were blocked with horse serum, incubated with an anti-PrP antibody (3F4), incubated with a biotinylated horse anti-mouse IgG secondary antibody using an ultrasensitive ABC peroxidase mouse IgG staining kit, and visualized with a 3,3′-diaminobenzidine (DAB) substrate kit (Thermo Fisher Scientific). After the sections were counterstained with hematoxylin, they were observed under a light microscope (BX51; Olympus, Southend-on-Sea, UK).