Ab186417

Immunohistochemistry was performed according to conventional procedures. Anti-SERPINA3 antibody (ab205198, Abcam, 1:2000) was used as the primary antibody, and goat anti-rabbit IgG was used as the secondary antibody to investigate SERPINA3 expression differences between patients with DN and healthy individuals. An anti-mast cell chymase antibody (ab186417, Abcam, 1:250) was used as the primary antibody, and goat anti-rabbit IgG was used as the secondary antibody to reveal the difference in chymase activity between patients with DN and healthy controls. Fluorescence colocalization staining was used to study the localization of chymase and SERPINA3, which were detected using the anti-SERPINA3 antibody (ab205198, Abcam, 1:2000) and anti-mast cell chymase antibody (ab186417, Abcam, 1:250), respectively. Images were collected with a confocal microscope (LeicaSP5-FCS, Wetzlar, Germany), and colocalization correlation analysis was performed using ImageJ software with the plugin Coloc 2 (18 (link)).
To detect the density of mast cells and the level of degranulation in the renal tubular tissues of patients with DN, tubule tissue sections were stained with toluidine blue (Servicebio, G1032). Ten non-overlapping regions were randomly selected for each section and analyzed by two observers. The total number of positive and granulated mast cells stained with toluidine blue was calculated.

Lung sections were deparaffinized and treated with antigen retriever (1X; Bio SB, Santa Barbara, California) for 5 min under microwave heating. Endogenous peroxidase was blocked incubating tissue with methanol-H2O2 (9:1) for 10 min. After three washes, unspecific sites were blocked using a background sniper (BIOCARE MEDICAL; Pacheco, California) for 30 min. Slides were washed and incubated with either a rabbit anti-human chymase antibody (Ab186417, Abcam; Cambridge, United Kingdom) or a mouse anti-tryptase antibody (Ab2378, Abcam; Cambridge, United Kingdom) for 2 h. After three washes, tissue was processed using a mouse/rabbit PolyDetector DAB (3–3′-diaminobenzidine)/HRP (horseradish peroxidase) brown detection system (BSB0219, Bio SB; Santa Barbara, California) following manufacturer´s instructions. Micrographs were acquired using a LEICA DMLS microscope with a 2.5X and 40X dry objectives equipped with a LEICA DFC295 camera and analysed using an automated image analyser (QWin Leica; Wetzlar, Germany).

At 24 hours after ICH, mice were perfused under deep anesthesia with cold PBS, followed by infusion of 4% paraformaldehyde^{59 (link)}. The brains were then removed and fixed in formalin at 4 °C overnight followed by dehydration with 30% sucrose in PBS. The frozen coronal slices (10 mm thick) were sectioned in cryostat (CM3050S; Leica Microsystems, Bannockburn, IL, USA). Brain slice were hydrated (30 minutes bi-distillated water room temperature) and stain with fresh prepared Toluidine Blue solution (0.1%, pH = 2.0) for three minutes. The slices were washed with distillated water three time, dehydrated through 75%, 95% and 2 changes of 100% alcohol, cleared in xylene substitute and coverslip with mounting medium. The perihematomal region of coronal brain sections were incubated overnight at 4 °C with the following primary antibodies: anti-tryptase 1:100 (Santa Cruz Biotechnology, sc-32889), anti-chymase 1:100 (Abcam, ab186417), anti-PIP3 1:100 (Abcam, ab11039), followed by incubation with appropriate FITC- conjugated secondary antibodies (Jackson ImmunoResearch). Sections were observed under an OLYMPUS BX51 microscope with fluorescence light.