Vectastain abc

IHC was performed with SOD-1, SOD-2, CAT and GPX to study changes in antioxidant immunoreactivities in the kidney. IHC was carried out according to our previously described method (22 (link)). In brief, the sections (6 µm) were incubated with primary goat anti-SOD1 (1:500; cat. no. SAB2500976; Sigma-Aldrich; Merck KGaA), goat anti-SOD2 (1:1,000; cat. no. SAB2501676; Sigma-Aldrich; Merck KGaA), rabbit anti-CAT (1:1,000; cat. no. ab16731; Abcam) and rabbit anti-GPX (1:1,000; cat. no. ab22604; Abcam) overnight at 4°C, followed by the biotinylated-conjugated anti-rabbit (1:250; cat. no. BA-1000-1.5; Vector Laboratories, Inc.) and the biotinylated-conjugated anti-goat (1:250; cat. no. BA-5000-1.5; Vector Laboratories, Inc.) secondary antibodies for 2 h at 24°C and developed using Vectastain ABC (Vector Laboratories, Inc.). Then, they were visualized with 3,3′-diaminobenzidine solution (in 0.1 M Tris-HCl buffer).
Leica DM 2500 microscope was used to image the sections at a magnification of ×400. A total of 10 sections/rat were selected and 10 areas were captured. ImageJ threshold analysis software version 1.52a (National Institutes of Health) was used to measure the percent (%) of relative optical density (ROD).

Lungs were inflation fixed at a constant pressure (25 cm H₂O) by tracheal installation of 4% paraformaldehyde, transferred to 70% ethanol after 24 h and embedded in paraffin as previously described (19 (link)). Immunostaining was performed on lung sections after antigen retrieval using Retrievagen A (Zymed, San Francisco, CA, USA) at 100°C for 20 min, and quenching endogenous peroxidases with 3% H₂O₂. Sections were blocked with 2% bovine serum albumin in PBS, followed by staining with primary anti-α-SMA (1:2,000; cat. no. 11475; BD Pharmingen, San Jose, CA, USA) at room temperature for 1 h. Sections were washed and after application of the goat HRP-conjugated anti-rabbit IgG secondary antibody (1:4,000; cat. no. HAF109; R&D Systems), tissues were developed using Vectastain ABC (Vector Labs, Burlingame, CA, USA) and 3,3′-diaminobenzidine (Vector Labs). After staining, five high-power fields (magnification, ×400) were randomly selected in each slide, and the average proportion of cells with positive expression in each field was counted using the true color multi-functional cell image analysis management system (Beckman Coulter, Inc, Fullerton, CA, USA), with values expressed as positive units (pu).

The lungs were inflated to a constant pressure of 25 cm H₂O. Then, 4%
paraformaldehyde was installed in the trachea. Subsequently, the lungs were
immersed in 70% ethanol for 24 h and paraffin-embedded.^{34 (link)} Following antigen retrieval with Retrievagen A (Zymed, South San
Francisco, CA, USA), the lung sections were immunostained for 20 min at 100°C,
and 3% hydrogen peroxide was used to quench endogenous peroxidases. Staining was
arrested by phosphate-buffered saline (PBS) containing 2% bovine serum albumin.
The sections were then stained with anti-α-SMA and HE4 antibodies, along with
primary anti-TG2 (BD Pharmingen, San Jose, CA, USA) for 1 h at room temperature.
The sections were rinsed and incu-bated with secondary antibodies (R&D
Systems, Minneapolis, MN, USA). Vectastain ABC (Vector Labs, Burlingame, CA,
USA) and 3,3′-diaminobenzidine (Vector Labs) were used for the development. Five
fields at high power (200×) were chosen on each slide following staining, and
the average size of the area of positive expression in every field was
determined using a true color multi-functional cell image analysis management
system (Image-Pro Plus; Media Cybernetics, Rockville, MD, USA). Values were
expressed as positive units.