Mouse anti α sma

Paraffin sections of iERM tissues were deparaffinized and hydrated through exposure with xylene and graded alcohols followed by water. As a pretreatment, microwave-based antigen retrieval was performed in 10 mM citrate buffer (pH 6). Sections were incubated with the following primary antibodies: rabbit anti-(P)RR (Sigma-Aldrich), mouse anti-prorenin, rabbit anti-ACE and rabbit anti-AT2R (Abcam, Cambrige, MA, USA), rabbit anti-AT1R and goat anti-AGT (Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-FGF2 (Millipore, Temecula, CA, USA), goat anti-GDNF, goat anti-NGF, mouse anti-TGF-β1 and mouse anti-α-SMA (R&D systems, Minneapolis, MN, USA), and mouse anti-GFAP (Leica, Exton, PA, USA) antibodies. Secondary antibodies for fluorescent detection were AlexaFluor 488 and 546 (Life Technologies). Sections were examined using the Keyence BZ-9000 (Keyence, Osaka, Japan).

Cell extracts were lysed in SDS buffer and a protease inhibitor cocktail (Promega). After quantifying protein concentrations using BCA reagent (Thermo Fisher Scientific), proteins were resolved by SDS-PAGE (polyacrylamide gel electrophoresis) and transferred to PVDF membrane by electroblotting. Membranes were blocked in TBS containing 5% skim milk, and probed with the following primary antibodies: mouse anti-α-SMA (1:2000, R&D Systems), rabbit anti-SM22 (1:2000), rabbit anti-type I collagen (1:2000), mouse anti-fibronectin (1:2000), mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:2000, Thermo Fisher Scientific), rabbit anti-SNAIL (1:1000, Cell signaling technology, Danvers, MA), and mouse anti-GS (1:1000, Millipore, Temecula, CA) antibodies. Horseradish peroxidase-conjugated anti-mouse and anti-rabbit IgGs (1:4000, Jackson ImmunoResearch Laboratories, West Grove, PA) were used as secondary antibodies for chemoluminescence detection. Signals were obtained by enhanced chemoluminescence (Perkin Elmer, Waltham, MA).

Immunofluorescence analyses were performed as described previously^{12 (link)}. Paraffin sections of iERM tissues were deparaffinized and hydrated through exposure with xylene and graded alcohols followed by water. As a pretreatment, microwave-based antigen retrieval was performed in 10 mM citrate buffer (pH 6). Sections were incubated with the following primary antibodies: rabbit anti-SNAIL (1:100, Proteintech, Rosemont, IL), mouse anti-TGF-β1 (1:50), mouse anti-S100 (1:50, Thermo Fisher Scientific), goat anti-TβRII (1:50), mouse anti-α-SMA (1:200, R&D Systems), goat anti-SM22 (1:100, Abcam, Cambridge, UK), mouse anti-GS (1:100, Millipore), and mouse anti-GFAP (1:100, Leica, Exton, PA) antibodies. Secondary antibodies for fluorescent detection were AlexaFluor 488 and 546 (1:400, Thermo Fisher Scientific). Nuclei were counterstained with DAPI, and sections were examined using a Biorevo BZ-9000 microscope (Keyence).