Ab190924

Cell or tissue extracts were prepared, and protein concentration was measured using the bicinchoninic acid assay. Primary antisera against KLK6 (ab190924), MMP9 (ab137867), and VEGFA (ab51745) were all purchased from Abcam (Cambridge, UK) and used at a dilution of 1:1,000. The secondary antiserum (Thermo Fisher Scientific, Waltham, MA, USA) was used according to the manufacturer's instructions at a dilution of 1:4,000. ECL substrate kit (ZSBio, Beijing, China) was used for the detection of proteins.

Immunohistochemistry was performed using the SP kit (SPN-9002) (ZSBio) according to the manufacturer's guidelines. Primary antibodies against KLK6 (ab190924), MMP9 (ab137867), and VEGFA (ab51745) were all purchased from Abcam, and used at a dilution of 1:50. The immunohistochemical images were quantified. Five fields of view were randomly selected on each section (with at least 200 cells per view), and the quantification was performed as follows:
Score A was assigned according to the proportion of positively-stained cells: 0 for <5%, 1 for 6%–25%, 2 for 26%–50%, 3 for 51%–75%, and 4 for >75%. An independent score B was assigned according to the intensity of staining: 0 for negative, 1 for moderate, 2 for strong, and 3 for very strong. A final score was then calculated according to the formula, score C = score A × score B. Eventually, samples with a score C above 5 were considered positive, while samples with a score C below 5 were considered negative.