Envision system horseradish peroxidase labeled polymer

Liver tissue samples were fixed in neutral buffered formalin (10%). Fixed samples were processed and stained with hematoxylin and eosin as described by Bancroft et al. (97 ). The immunohistochemical protocol was conducted following the method of Saber et al. (98 (link)). Wax was removed from tissue sections, and they were rinsed in 0.05 M citrate buffer, pH 6.8. Non-specific binding was blocked by treating the sections with 0.3% H₂O₂ and a protein block. Sections were subjected to rabbit monoclonal (anti-bcl2, Abcam, Cat# ab182858, dilution 1:500) primary antibody. Sections were washed in phosphate-buffered saline and subjected to goat antirabbit secondary antibody (EnVision System Horseradish Peroxidase Labeled Polymer; Dako) for 30 min at room temperature. Slides were visualized with DAB kit and counterstained using Mayer's hematoxylin. The immunolabeling indices of Bcl2 are presented as a percentage of positive expression in a total of 1,000 cells per eight high power fields (HPF).

For immunofluorescence analysis, the paraffin tissue sections were cut, mounted, deparaffinized, and rehydrated. After blocking, the sections were incubated with primary antibodies against CD45 and Mac2 (Abcam; Cambridge, UK) in 3% BSA overnight at 4 °C, followed by incubation with the corresponding secondary antibody for 1 h at 25 °C, and nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). The CD45- and Mac2-stained cells within the neointima were directly counted in four high-power fields, and the mean numbers of cells were then compared [38 (link)]. For immunohistochemical staining, primary antibodies against MMP-9 (Sigma-Aldrich) and α-SMA (Abcam) were used. After overnight incubation, the tissue sections were washed and incubated using EnVision system-horseradish peroxidase-labeled polymer (Dako; Glostrup, Denmark) for 1 h at room temperature. The sections were visualized with 3,3′-diaminobenzidine tetrahydrochloride (Dako) and counterstained with hematoxylin. The MMP-9-stained cells were counted in four high-power fields and compared. The α-SMA-stained area in neointima was analyzed using ImageJ software and expressed as ratio of positive area as Picro Sirius red stain. For both immuno-staining examinations, three sections in the most severe neointimal hyperplasia region, 200 μm apart, were used for each staining.