Anti arginase 1 arg 1

Total protein extracts were obtained after washing the hearts with PBS and adding 300 ml of RIPA modified lysis buffer (50 mM NaCl, 50 mM Tris–HCl (pH 7.40), 1% Triton X-100, 1 mM EDTA, 1 mM PMSF; 2.5 g/l Protease Inhibitor Cocktail (Sigma-Aldrich Co., St. Louis, MO, USA), 1 mM Na₃VO₄, 1 mM NaF), or washing the cultured cells and scraped off the dishes with 50 µl of the same buffer. Then, the tubes were kept on ice for 30 min with swirling, and the samples were centrifuged at 7,000 g at 4°C for 10 min. The supernatants were stored at −20°C. Protein concentrations were determined by the Bradford method using the Bio-Rad Protein Assay (Bio-Rad, USA) and bovine serum albumin (Sigma-Aldrich Co., St. Louis, MO, USA) as a standard (36 (link)). For Western blot analysis, total proteins were boiled in Laemmli sample buffer, and equal amounts of protein (40–50 µg) were separated by 10–12% SDS-PAGE. The gels were blotted onto a Hybond-P membrane (GE Healthcare, Madrid, Spain) and incubated with the following antibodies: anti-NOS2, anti-NOS3, anti-Arginase I (Arg-I), anti-CD31, anti-VEGF-A, and anti-α-actin (Santa Cruz Biotechnology, CA, USA). The blots were revealed by enhanced chemiluminescence in an Image Quant 300 cabinet (GE Healthcare Biosciences, PA, USA) following the manufacturer’s instructions. Band intensity was analyzed using the NIH-ImageJ software (37 (link)).

Sections were deparaffinized and rehydrated prior to antigen retrieval as described (23 (link)). Tissue sections were then incubated overnight at 4°C with 1:50 dilution of anti-arginase I (ArgI, Santa Cruz Biotechnology) primary antibody followed by 1:1000 dilution of Alexa 568-conjugated anti-goat secondary antibody. A mixture of anti-NOS2 (BD Transduction Labs; 1:50 dilution) and anti-F4/80 (ABD-Serotec; 1:50 dilution) primary antibodies were then applied for 1 h at 37°C, followed by 20 min incubations with Alexa 488-conjugated anti-rabbit and Alexa 680-conjugated anti-rat secondaries. Nuclei were stained with DAPI-containing mounting media (Vector Laboratories). Images were obtained with a digital deconvolution microscopy imaging system attached to a Zeiss Axioplan 2 epi-Fluorescence upright microscope. Macrophages were identified by positive F4/80 staining and morphology. Total pixel counts/macrophage were calculated for ArgI, NOS2, and F4/80 immunofluorescence (~50/animal) using ImageJ software (38 (link)), and ArgI and NOS2 values were normalized to F4/80 staining. To confirm ArgI⁺ M2 programing, adjacent sections were subjected to a similar IF protocol substituting an antibody against M2 marker phosphoTyr⁶⁴¹STAT6 (Cell Signaling, 1:50 dilution) for NOS2. Fluorescence intensity was calculated similarly and phosphoSTAT6/ArgI ratios determined.