Mouse anti mast cell tryptase antibody

Immunohistochemistry (IHC) stains were used to evaluate the expression of major basic protein (MBP) and tryptase, which are markers of eosinophil and mast cell degranulation, respectively26 (link). After dewaxing and rehydrating, tissue sections (D1, D2) were treated with 0.3% hydrogen peroxide in methanol to block endogenous peroxidase activity followed by pepsin or heat-mediated antigen retrieval. After blocking with 3% goat serum for 20 minutes at room temperature to minimize nonspecific staining, the sections were incubated with mouse anti-eosinophil MBP antibody (1:50, AbD Serotec, UK) and mouse anti-mast cell tryptase antibody (1:26000, Abcam, USA) for 1 hour at room temperature. After washing, the sections were treated with HRP-labeled goat anti-mouse IgG (ZSGB, China) for 30 minutes. The reaction was visualized using diaminobenzidine (DAB kit, ZSGB, China) and hematoxylin staining. Eosinophil degranulation positive was defined by the presence of MBP granules stain in randomly selected fields. If there was evidence of eosinophil degranulation on IHC stain, five non-overlapping fields on the slides (400× magnification, Nikon) were randomly selected and analyzed by two independent observers (LD, LM), to determine the mean numbers of degranulated eosinophil and mast cell and expressed per HPF.

RBL cells were seeded on 10 mm diameter coverslips and kept for adherence at 37 °C in CO₂ incubator. CFSE labelled L. tropica and L. donovani were added to the cells at MOI 1:10 for 24 h. The medium was removed and cells were fixed with 2% PFA in PBS for 30 min and excess PFA quenched with 50 mM NH₄Cl in PBS. The PFA fixed cells were then incubated with 3% normal goat serum (Sigma, MO, USA) for 1 h to prevent nonspecific protein binding. Rabbit Anti-Histone H2A (acetyl K5) antibody (Abcam, Cambridge, MA) and Mouse anti-Mast Cell Tryptase antibody (Abcam, Cambridge, MA) diluted in the same buffer were added to the cells, and incubation was conducted for 2 h. After washing, this was followed by 30-min incubation in the presence of secondary goat Abs conjugated to Alexa Fluor 546 (red). Coverslips were mounted in Vecta shield containing DAPI (Vector, Vector Lab. Inc.) to stain nucleus. The images were visualized under Nikon Real Time Laser Scanning Confocal Microscope Model A1R with motorized inverted microscope having Live Cell and Spectral Imaging – Model Ti-E at 60X.

BMMCs were plated to the slide and were fixed by heating with 10% (w/v) neutral buffered formalin solution. Cells were reacted with mouse anti-mast cell tryptase antibody (AB2378, Abcam, Cambridge, UK). Cells were then reacted with FITC-conjugated anti-mouse IgG (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and 1µg/mL DAPI (4′,6-diamidino-2-phenylindole dihydrochloride). Paraffin sections were reacted with respective antibodies and analyzed as described previously [53 (link)]. Sections were subjected to immunohistochemistry using antibodies of Alexa Fluor 488^®-anti-mouse Il-10 (#505013), Brilliant Violet 421™-anti-mouse TGF-β1 (#141407), Pacific Blue™-anti-mouse IL-2 (#503820), Alexa Fluor^® 488-anti-GATA3 (#653808), PE-anti-mouse Ly-6A/E (Sca-1) (#108108), PE-anti-mouse IL-33Rα (ST2) (#146608) and PE-anti-mouse/human IL-5 (#504304) (BioLegend, San Diego, CA, USA), FITC-anti-mouse/human CD45R (#11-0452-82) (Thermo Fisher Scientific, Waltham, MA, USA), PE-anti-mouse CD25 (#130-120-766) (Miltenyi Biotech, Bergisch Gladbach, Germany). All antibodies were used at a 1:500 dilution.