Anti fga

Mouse tissues were lysed using a protein lysis buffer containing 20 mM Tris (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 25 mM sodium pyrophosphate, and 2 mM sodium orthovanadate aprotinin. All denatured proteins were separated on an SDS-PAGE gel and then transferred to polyvinylidene difluoride membranes (Roche, Netley, NJ, United States). The membranes were blocked with 5% skimmed milk in Tris-buffered saline and then were incubated with 1:500 dilutions of primary antibodies as follows: anti-FGA (Abcam, Cambridge, MA, United States), anti-Slc12a1 (Abcam, Cambridge, MA, United States), and anti-Havcr1 (Abcam, Cambridge, MA, United States). Then, the samples were incubated with a horseradish peroxidase-conjugated anti-rabbit secondary antibody (Jackson ImmunoResearch, PA, United States). The bands were visualized using an ECL Western Blotting Kit (Biovision, Milpitas, CA, United States) and were quantified by Quantity One software (Bio-Rad, Hercules, CA, United States).

Immunohistochemical staining for TSP1, AHSG and FGA expression was performed on multi-tissue microarrays (MTAs) (Outdo Biotech, Shanghai, China). Tissues were stained with anti-TSP1 (Proteintech Group, IL), anti-AHSG (Sigma-Aldrich, MO) and anti-FGA (Abcam, UK) antibodies, and images were captured using Aperio ScanScope CS software (Vista). After calculating the informative cases, the intensity of staining in individual cases was quantified as previously described [37 (link)]. A score of 4–12 was defined as positive expression, and a score of 0–3 was considered negative.