Immunohistochemistry (IHC) for assessment of matrix metalloproteinase-9 (MMP-9), (FS7-associated cell surface antigen) FAS protein, caspase-3, BAX, and BCL2 expression

We utilized the paraffin specimens of formalin-fixed renal samples for immunohistochemical examination. Paraffin sections of 4 μm thickness were prepared and mounted on positively charged slides, deparaffinized in xylene, hydrated in descendant ratings of ethanol, and treated with 3% hydrogen peroxide in methanol to block the endogenous peroxidase action. Antigens were retrieved using 0.01 mol/L citrate-buffered saline (pH 6.0), and endogenous peroxidase activity was quenched using 0.3% (v/v) H₂O₂ in phosphate-buffered saline. Then, the non-specific binding of immunological reagents was blocked by incubating the samples with normal goat serum 10% (v/v) for one hour. A goat anti-mouse MMP-9 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) diluted 1:100 was applied to ultrathin sections overnight at 4 °C, followed by a second layer of biotin-conjugated, affinity purified rabbit anti-goat immunoglobulin G (Santa Cruz) diluted 1:100 with phosphate-buffered saline and 0.1% bovine serum albumin for 15 min at room temperature, and finally by streptavidin-conjugated horseradish peroxidase, rabbit monoclonal FAS antibody (ab-15285 Abcam, Cambridge, UK) with a dilution of 1:100 in PBS for 1 h at room temperature and anti-BAX, rat monoclonal antibody (1:50, no. 13401A, clone G206-1276, immunoglobulin (Ig) M, 0.5 mg/mL, PharMingen, San Diego, CA). Additionally, anti-active caspase-3 antibody incubation (Abcam, Cambridge, MA, USA) was used. Anti-active caspase-3 antibody was diluted to 1:300. Moreover, monoclonal mouse using a Bio-Rad GS-690 densitometer and Molecular anti-rat Bcl-2 (to confirm Bcl-2 staining; Santa Cruz BioAnalyst version 4 analysis software) (Bio-Rad Laboratories chemicals) were also used. Polyclonal mega using the same analysis package and confirmed rabbit anti-rat Bcl-2 (1:400; PharMingen, San Diego, CA, by visual comparison to the ribosomal RNA subunits, USA) was diluted at 1:400. The primary antibodies were detected with a biotin-streptavidin detection system with 3,3′-Diaminobenzidine (DAB) (Sigma Aldrich, USA) as a chromogen, and then, counter-staining with Mayer’s hematoxylin was performed. Negative control segments were prepared with similar rules with replacement of the definite primary antibodies with normal rabbit immunoglobulin G (IgG) (Suvarna et al. 2018).