Vectastain elite abc staining kit

Sections (5 μm) of formalin-fixed tissue sections were stained with hematoxylin and eosin according to standard procedures. Sections were incubated in 0.3% H₂O₂, and followed by another 30 min in 1% BSA. Then, sections were incubated with anti-myeloperoxidase (MPO) (Biocare Medical, USA) primary antibodies overnight at 4°C. Vectastain Elite ABC Staining Kit and DAB Peroxidase Substrate Kit (Vector Laboratories, USA) were used to visualize the staining according to the manufacturer's instructions.

Paraffin-embedded tissues were deparaffinized and subjected to antigen retrieval with citrate buffer pH 6.0 and treated with 3% hydrogen peroxide to block endogenous peroxidase activity. Tissues were blocked with serum and single staining was done by incubating primary antibody Lon (LSBio #LS-C752623) or NCLX (Proteintech #21430-1-AP) at 4 ^oC overnight. Slides were stained with biotin-conjugated antibody, streptavidin peroxidase (VECTA STAIN Elite ABC staining kit, VECTOR laboratories, USA) followed by substrate addition (VECTOR NovaRED substrate kit, peroxidase VECTOR laboratories, USA) to observe immnoreactivity. Slides were scanned using PANNORAMIC Digital slide scanner.

Formalin-fixed liver samples were processed, and 4 µm thick paraffin sections were stained with Sirius Red dye (Sigma-Aldrich, St. Louis, MO, USA). For hematoxylin and eosin staining, paraffin-embedded sections were stained with hematoxylin and eosin (Thermo Fisher Scientific, Waltham, MA, USA). TUNEL staining was performed using an ApopTag^® Peroxidase In Situ Apoptosis Detection Kit (Millipore, Burlington, MA, USA). For immunohistochemistry, after heat-induced epitope retrieval, paraffin-embedded sections were incubated in 3% H₂O₂ and blocked in 3% normal serum buffer. The sections were incubated with primary antibodies overnight at 4 °C. The Vectastain Elite ABC staining kit and DAB peroxidase substrate kit (Vector Laboratories, Burlingame, CA, USA) were used to visualize the staining according to the manufacturer’s instructions. Immunohistochemical assessment was performed using primary antibodies against MPO (Biocare Medical, Concord, CA, USA), Ly6G (BioXCell, West Lebanon, NH, USA), MDA (Genox, Baltimore, MD, USA), 4-HNE (Genox), F4/80 (Cell Signaling Technology, Danvers, MA, USA), and α-SMA (Agilent DAKO, Santa Clara, CA, USA). Subsequently, we analyzed the positive cells and positive areas in 10 randomly selected high-power fields. The concentrations of antibodies used are described in Supplementary Table S2.

Four-micron thick paraffin sections of kidneys were deparaffinised, rehydrated and rinsed. Heat-mediated antigen retrieval method with sodium citrate buffer (10 mM sodium citrate, 0.05% tween 20, pH 6.0) was performed. Sections were then quenched with 3% H₂0₂ for 20 minutes and incubated with 10% (rabbit/goat) serum for 30 minutes to block non-specific binding. Sections were incubated with rat monoclonal to IL-10 (ab189392; Abcam, Cambridge, UK) or rabbit polyclonal to PAI-1 (ab66705; Abcam, Cambridge, UK) antibody (1:100) overnight at 4 °C. The next day, sections were washed and incubated with anti-rat/anti-rabbit secondary antibody (1:200) for 30 minutes at room temperature. This was followed by incubation with horseradish peroxidase-conjugated streptavidin (VECTASTAIN Elite ABC staining kit; Vector Laboratories). 3,3′-diaminobenzidine tetrahydrochloride was then used to visualise peroxidase conjugates in samples. All slides were then hydrated, cleared and mounted with DPX. Ten random fields per animal were imaged in the renal cortical region using the Olympus BX43 microscope (x20 magnification). Staining was quantified by counting the number of positively stained cells per field. All assessments were performed in a blinded manner.