Sc 18811

Detailed protocols used for histological and immunohistochemical staining have been previously described55 (link). For Masson’s trichrome staining the hearts were fixed in 4% paraformaldehyde, dehydrated through an ethanol series, embedded in paraffin, sectioned and then stained. Immunohistochemistry was also performed on fixed, paraffin embedded sections with rabbit anti-ANP antibodies (sc-18811, Santa Cruz Biotechnology). Primary antibodies were recognized by Alexa488 goat anti-rabbit antibodies (Jackson ImmunoResearch Laboratories).

Lung and heart tissues were lysed with a buffer containing RIPA (Beyotime, China), 10 % phosphatase inhibitor (Roche Applied Science, Germany) and 1 % proteinase inhibitor (Sigma, USA). Bicinchoninic acid assay was used to measure the protein concentration. Equal amounts of the samples were loaded on 10 % SDS–PAGE gels. Then, protein was transferred to a PVDF membrane (Millipore Corporation, USA) and incubated for 1 h at room temperature in blocking solution (5 % non-fat milk). The membrane were incubated overnight at 4 °C in blocking solution containing primary antibodies. Then, it was washed and incubated with horseradish peroxidase conjugated secondary antibody (Cell Signaling Technology, USA) for 1 h at room temperature. After a second wash, the membrane was developed using enhanced chemiluminescence substrate (Millipore Corporation, USA). The band intensities were analyzed using Image J software (National Institutes of Health, Bethesda, USA). Primary antibodies against renin, Ang II, ACE, mineralocorticoid receptor (MR), BNP, phospho-ERK1/2 (Thr202 + Tyr204) and β-actin were purchased from BIOSS Biotechnology (bs-6184R, bs-0587R, bs-0439R, bs-1850R, bs-7132R, bs-3016R, bs-0061R). Primary antibodies against Ang II type 2 receptor (AT2 receptor) and ANP were obtained from Santa Cruz Biotechnology (sc-9040, sc-18811).