Baf1097

Cultured macrophages were lysed on ice with cold radio immunoprecipitation assay (RIPA) lysis buffer (in house) supplemented with protease inhibitors (Complete protease inhibitor cocktail tablets, Roche Diagnostics, #11697498001) and protein concentration was measured using BCA protein assay (Pierce BCA Protein Assay Kit, #23225, ThermoFisher Scientific). Equal amounts of protein were loaded onto 10% SDS-polyacrylamide gel and transferred to an Immobilon-P transfer membrane (# IPVH00010, PVDF membrane, Millipore). The blots were blocked for 1 h using 10% milk in Tris-Buffered Saline and 0.1% Tween-20 solution and incubated O/N with goat anti-mouse endoglin (1:1000 dilution, BAF1097, R&D Systems) or mouse anti-β-Actin (1:10.000 dilution, A5441, Sigma-Aldrich). Blots were incubated for 1 h with horse radish peroxidase anti-goat (goat anti-mouse IgG Poly-HRP Secondary Antibody HRP conjugate, #32230, ThermoFisher Scientific) or anti-mouse (ECL mouse IgG, HRP-linked whole Ab #NA931, Sigma-Aldrich, GE Healthcare, UK). Blots were developed in a Kodak X-omat 1000 processor with Thermo Scientific SuperSignal West Dura (Extended Duration Substrate) or SuperSignal West Pico and exposed to Fuji SuperRX medical X-ray film. Analysis was performed using Image J (National Institute of Mental Health, Bethesda, Maryland, USA).

Immunohistochemistry was performed as described before (23 (link)). In short, FFPE sections (4 μm) were deparaffinized, blocked in 0.3% hydrogen peroxide (H₂O₂) in methanol for 20 min, and rehydrated. Antigen retrieval was performed by boiling the sections in a 0.01 M citrate solution (pH 6.0) for 10 min. After being washed with phosphate-buffered saline (PBS), the sections were incubated with polyclonal goat anti-human endoglin (1:400—BAF1097, R&D systems, MN, United States) in PBS/1% bovine serum albumin (BSA), and left overnight at room temperature. The next day, the slides were washed and then incubated with a biotinylated polyclonal rabbit anti goat secondary antibody (Dako, CA, United States) for 30 min, washed and incubated with Vectastain complex (Vector Laboratories, CA, United States). The color was developed using a 3,3’diaminobenzidine (DAB) + substrate chromogen system (Dako, CA, United States), following the manufacturer instructions. Nuclear staining was performed using hematoxylin (Merck, Darmstadt, Germany). Slides were dehydrated and mounted using entellan (Merck, Darmstadt, Germany).