Ab 151757

For immunofluorescence, 3 μm thick tissue sections adhered to silanized slides (Sigma-Aldrich, St. Louis MO, USA) were used. After heat-induced antigen retrieval in citrate buffer, samples were permeabilized and blocked in a single step incubating them for 30 minutes in PBS with 2% normal goat/porcine serum and 0.5% Triton X-100 (Research Organic, Cleveland, OH, USA) at room temperature (RT). Afterwards samples were washed and incubated at 4ºC overnight with primary antibodies: anti-TMPRSS4 [Santa Cruz Biotechnology/ sc-376415 (1:100)], anti-Tryptase [Abcam/ ab-151757 (1:250)] and anti-Chymase [Abcam/ ab-111239 (1:300)]. The next day samples were washed and incubated for 1 hour at RT with fluorescent secondary antibodies (Alexa Fluor[AF]-488 conjugated donkey anti-Rabbit IgG, Dylight-549 conjugated donkey anti-mouse IgG and AF-647 conjugated donkey anti-goat IgG; (Jackson Immunoresearch, West Grove, PA). Nuclei were stained with NucBlue (Life Technologies, Carlsbad, CA). Samples were mounted with ProLong Gold mounting media (Life Technologies). All experiments included controls with/without primary antibody or with/without secondary antibody. Imaging was performed with an FV-1000 confocal laser scanning microscope (Olympus Corporation, Tokyo, Japan) in sequential scanning mode to image each fluorochrome separately.