Anti arginase 1 h 52

Mouse tissues were homogenized in ice‐cold lysis buffer containing T‐PER (Thermo Scientific, Waltham, MA), 1 mmol/L sodium fluoride (Sigma‐Aldrich), 1 mmol/L sodium orthovanadate (Sigma‐Aldrich), complete protease inhibitor cocktail (Roche Applied Science, Penzberg, Germany) and 1 mmol/L phenylmethanesulfonyl fluoride (Sigma‐Aldrich). Samples were loaded onto 10% SDS gel, separated by electrophoresis, and transferred to PVDF membranes. Membranes were then incubated overnight with primary antibodies at 4°C. Primary antibodies used were as follows: anti‐arginase 1 (H‐52; Santa Cruz Biotechnology, Dallas, TX), anti‐arginase 2 (H‐64; Santa Cruz Biotechnology), anti‐β‐actin (4967; Cell Signaling Technology, Beverly, MA), anti‐caspase‐3 (H‐277; Santa Cruz Biotechnology), and anti‐GAPDH (MCA2427; AbD). After incubation with HRP‐conjugated secondary antibody and SuperSignal West Pico Chemiluminescent Substrate (Pierce, Waltham, MA), signals were visualized using LAS‐3000 mini (Fujifilm, Tokyo, Japan). The relative expression levels to β‐actin (n = 5) were quantified by the densitometric analysis using ImageJ 1.42q (National Institute of Health, U.S.A.).

Mice were sacrificed and perfused with PBS and 4% paraformaldehyde: organs were immediately frozen in O.C.T. compound (Sakura Finetek Japan, Tokyo, Japan) or fixed overnight at 4°C and embedded in paraffin. Immunohistochemistry, immunofluorescence staining, and Masson's trichrome staining were performed as previously described (Hakuno et al. 2010 (link)). The following primary antibodies were used: anti‐arginase 1 (H‐52; Santa Cruz Biotechnology), anti‐arginase 2 (H‐64; Santa Cruz Biotechnology), anti‐α‐smooth muscle actin (SMA) (A5228, Sigma‐Aldrich), and anti‐Mac‐3 (BD Biosciences, San Jose, CA). Secondary antibodies for immunofluorescence staining were Alexa Fluor 546 and Alexa Fluor 488 goat IgG (Molecular Probes, Carlsbad, CA). The nuclei were stained with DAPI (Molecular Probes). The slides were observed under a microscope (AX80N‐65; OLYMPUS, Tokyo, Japan) or an immunofluorescence microscope (Biozero BZ‐8100; Keyence, Osaka, Japan). Cell size distribution of cardiomyocyte was assessed by measuring cross‐sectional area of 50 cardiomyocytes having nearly circular capillary profiles in the LV free wall. Histological images were analyzed by using ImageJ 1.42q.