Vactastain abc peroxidase kit

Detection of proliferating cell nuclear antigen (PCNA) expression on kidney’s paraffin sections of selected control and treated rats using avidin-biotin Peroxidase (DAB, Sigma Chemical Co.) was done according to method described by [23 (link)]. Tissue sections were incubated with a monoclonal antibody to PCNA (Dako Corp, Carpenteria, CA) and reagents required for the avidin-biotin peroxidase (Vactastain ABC peroxidase kit, Vector Laboratories) method for the detection of the antigen—antibody complex. PCNA expression was localized by the chromagen 3,3-diaminobenzidine tetrahydrochloride (DAB, Sigma Chemical Co.).

Immunohistochemistry for MMP-9 and NF-κB p65 expression was formed using the avidin-biotin-peroxidase technique. Paraffin sections were de-waxed and dehydrated, followed by heat treatment for antigen retrieval. Sections were incubated with monoclonal antibodies of MMP-9 and NF-κB p65 (Dako Corp, Carpenteria, Santa Clara, CA 95051, USA) at dilutions of (1:200 and 1:100, respectively) as well as the other reagents adopted for the avidin-biotin-peroxidase technique (Vactastain ABC peroxidase kit, Vector Laboratories, Burlingame, CA 94010, USA) according to methods mentioned by [38 (link)]. Visualization of each marker expression was achieved by Chromagen 3,3 -diaminobenzidine tetrahydrochloride (DAB, Sigma Chemical Co., Merck, Darmstadt, Germany). Image analysis software (Image J, 1.46a, NIH, USA) was used for quantitative analysis of the immune stained sections by measuring the optical density of the positive brown color in 5 microscopic fields (the area for each microscopic field is 18.893 Sqmm).

The other paraffin section from each group was used for immunohistochemical detection of the expression of PERK in various experimental groups using avidin–biotin peroxidase according to method described by^{32 (link)}. To detect antigen–antibody complexes, kidney slices were treated with monoclonal antibodies for PERK (Abcam, Cambridge, MA, USA) at a dilution of 1:200 (Vactastain ABC peroxidase kit, Vector Laboratories). Chromagen diaminobenzidine tetrahydrochloride was used to visualize each marker expression (DAB, Sigma-Aldrich, St. Louis, MO, USA). The positive brown area of each marker expression was measured using Image J, 1.46a analysis software (NIH, USA), and seven high-power microscopic fields.

The other paraffin section from each group was used for immunohistochemical detection of the expression of p‐AKT in various experimental groups using avidin‐biotin‐peroxidase according to the method described by.
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For the purpose of identifying a bound antigen and antibody, liver cuts were treated with antibodies for p‐AKT (Abcam, Cambridge, MA, USA) at a dilution of 1:200 (v/v) and (Vactastain ABC peroxidase kit, Vector Laboratories, Burlingame, USA). Chromagen 3, 3‐diaminobenzidine tetrahydrochloride was used to visualize each marker's expression (DAB, St Louis, MO, USA).

The other paraffin section from each group was used for immunohistochemical detection of the expression of Stat3 in various experimental groups using avidin-biotin-peroxidase according to the method described in [33 (link)]. Kidney slices were treated with monoclonal antibodies for Stat3 (Abcam, Cambridge, MA, USA) at a dilution of 1 : 200 and (Vactastain ABC peroxidase kit, Vector Laboratories, Newark, USA) for the aim of identifying antigen-antibody complexes. Each marker's expression was visualized using chromagen 3,3-diaminobenzidine tetrahydrochloride (DAB, Sigma Chemical Co.). The positive brown region of each marker's expression was assessed using seven high-power microscopic fields and image analysis software (ImageJ, 1.46a, NIH, Bethesda, USA).

According to the procedure outlined by Hsu et al., avidin-biotin-peroxidase and diaminobenzidine tetrahydrochloride DAB, Sigma-Aldrich (St. Louis, MO, USA) was used in immunohistochemical experiments to identify caspase-3 and α-SMA expression on paraffin slices of the control liver and all treatment groups [22 (link)]. For the avidin-biotin-peroxidase technique of detecting the antigen-antibody complex, tissue slices were incubated with a monoclonal antibody for caspase-3 and α-SMA (Dako Corp, Carpenteria, CA, USA) and the necessary chemicals (Vactastain ABC peroxidase kit, Vector Laboratories, Newark, USA). Using DAB, each marker expression was seen.