Chemmate envision detection kit

The ChemMate EnVision Detection Kit (Dako, Carpinteria, CA, USA) and paraffin blocks of colorectal cancer tissue samples and normal colon mucosa biopsy samples were used for immunohistochemical analysis. Briefly, the paraffin-embedded samples were deparaffinized in xylene and dehydrated with ethanol. For antigen retrieval, the paraffin-embedded sections were placed in 0.01 M sodium citrate buffer (pH 6.0) and subjected to pressure cooker treatment for 5 min at full pressure. After cooling to room temperature, the sections were incubated with 3% hydrogen peroxide to block endogenous peroxidase activity. Then, nonspecific antigens were blocked with 10% goat serum (100 μL goat serum in 900 μL PBS) at room temperature for 30 min. The sections were incubated with the primary antibody (1:200 dilution) overnight at 4°C. The primary antibody used in the present study was a rabbit polyclonal antibody against the human SUPT5H protein (HPA029273, Sigma, CA, USA). Then, the ChemMate EnVision/HRP, Rabbit/Mouse (ENV) reagent was applied to the sections, followed by application of ChemMate DAB+ Chromogen, which was included in the kit. The sections were counterstained with hematoxylin for 3 min and then were rinsed, dehydrated, and covered with a glass slip.

Tumor tissues and organs were resected from mice and fixed with formalin and embedded in paraffin and then prepared as 3-mm-thick sections. The organ slides were directly stained with HE. The tumor tissue sections were stained for the presence of human T cells using a mouse monoclonal anti-human CD3ε antibody (Thermo Scientific) and the proliferation of tumor cells using a mouse anti-human Ki67 antibody (Abcam). Following incubation with the primary antibody overnight at 4°C, the secondary antibody was added and the results were visualized using a ChemMate Envision Detection Kit (DakoCytomation).

Paraffin-embedded carotid arteries were deparaffinized in xylene, rehydrated in graded ethanol, and subjected to immunostaining. Immunohistochemistry was performed on serial sections, as described previously [9 (link)]. The primary antibodies used were macrophage marker CD68 clone PG-M1 (Dako, Glostrup, Denmark), smooth muscle actin (SMC) clone 1A4 (Dako), ferritin, TfR (Dako), and thrombin receptor (protease-activated receptor 1, Sigma, Saint Louis, USA). The immunoreactions were visualized using the EnVision+/horseradish peroxidase (Dako) method and ChemMate EnVision Detection Kit (Dako). Control sections without primary antibodies or with non-immune IgG were run for each protocol, resulting in consistently negative results. The slides were counterstained with hematoxylin.
All histological sections were examined under a light microscope, and the images were digitalized with the Image Grabber program (Toronto, ON, Canada). The microscope was set on the same parameters used to scan all samples. The randomly digitalized images were analyzed with Adobe Photoshop (v5.5) as described previously [9 (link)]. The individual responsible for the analysis was blinded to patient information.