Mouse anti sma

Cells and tissues were fixed in PFA 2% and processed for histology and immunocytochemistry as previously described (Gargioli et al, 2008 (link)). The primary antibodies used were mouse anti-Pax7 (DSHB) at 1:20, mouse MF20 (DSHB) at 1:2, rabbit anti-laminin (SIGMA #9393) at 1:100, rabbit anti-LacZ (Cappel) at 1:100, rat anti-VE-cadherin (clone BV13 homemade) at 1:100, mouse anti-SMA (Sigma) at 1:100, mouse anti-dystrophin (Vector) at 1:100, mouse anti-neuronal class III β-tubulin (COVANCE) and alpha-bungarotoxin Alexa594 (Molecular probes) at 50 mg/ml. The secondary antibodies used at 1:100 were anti-mouse Alexa555 (Molecular Probes), anti-rabbit Alexa488 (Molecular Probes) and anti-rat Alexa568 (Molecular Probes), Cy2-anti-mouse (Jackson), AMCA-anti-mouse (Jackson), goat anti-mouse horseradish peroxidase (HRP)-conjugated IgG (Bio-Rad) for immunohistochemistry against MF20, developing the peroxidase reaction by AEC (3-amino-9-ethylcarbazole) substrate (SIGMA). The sections were photographed with Nikon ECLIPSE 2000-TE microscope or with Olympus FV 1000 confocal laser scanning microscope for the confocal images. VE-cad-positive capillary endothelial cells were counted under fluorescence microscopy (×200) in five randomly selected fields of different sections from each sample and related to the number of muscle fibres in the same section (Díaz-Manera et al, 2010 (link)).

Total protein extracts were prepared as previously described^{33 (link)}. The membranes were probed with the following antibodies: rabbit anti-LC3B (1:4000; Sigma), mouse anti-ATG5 (1:1000; Sigma), rabbit anti-ATG7 (1:4000; Sigma), rabbit anti-p62/SQSTM1 (1:4000; Sigma), rabbit anti-collagen (1:1000; Abcam), mouse anti-SMA (1:4000; Sigma), mouse anti-vimentin (1:4000; Sigma), mouse anti-β-actin (1:10000; Novus), rabbit anti-phospho-Smad2/3 (1:1000; Cell Signaling) and rabbit anti-Smad2/3 (1:1000; Cell Signaling). After an overnight incubation with each primary antibody, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody. The proteins were then detected using an enhanced chemiluminescence detection system (Thermo). Densitometric analysis was performed using Image J software (NIH). Relative intensities were calculated by normalizing the intensities of each marker to the loading control.

Lung tissues or cells were lysed in RIPA buffer containing complete protease inhibitor and PhosSTOP phosphatase inhibitor cocktails (Roche Applied Science). Lysate was centrifuged at 13,500 × g for 10 min at 4 °C, and the supernatant was collected. Protein concentrations were determined by the BCA assay (Pierce). Laemmli buffer (Bio-Rad) containing β-mercaptoethanol was added to 40 μg protein, and boiled at 95 °C for 5 min. Protein were separated by SDS–PAGE, transferred to nitrocellulose membranes (Millipore) overnight, blocked with 5% bovine serum albumin in PBS containing 0.05% Tween-20 for 1 h and incubated with primary antibody overnight at 4 °C. Primary antibodies used were: rabbit anti-KLF4, rabbit anti-SMAD2/3, rabbit anti-GAPDH (1:500, 1:500, 1:5000; Cell Signaling Technology), rabbit anti-phospho-SMAD3, rabbit anti-collagen 1, rabbit anti-CCL2, rabbit anti-FOXM1 (1:500, 1:1000, 1:1000, 1:1000; Abcam), mouse anti-SMA (1:2000, Sigma) and mouse anti-fibronectin (1:5000, BD Biosciences). After washing, membranes were incubated with horseradish peroxidase–conjugated secondary antibodies (1:2000; DAKO) for 1 h. Detection was performed with the Western Blotting Substrate Plus (Pierce) and GBOX imaging system (Syngene).

Cells were formalin fixed, permeabilized and stained with rabbit anti-NHE1 antibody (United States Biologicals, N2015-06.100) conjugated to goat anti-rabbit Alexa 488 (green), mouse anti-SMA (Sigma, A2547) conjugated to goat anti-mouse CY3 (red). In some experiments, the nuclear stain YO-PRO (green) was added to help visualize cells. FRET efficiency was performed using confocal microscopy and standard protocols. Cells were excited at 488 nM and emitted fluorescence for each fluorescent stain was measured, followed by photobleaching (30 scans at 100% lamp intensity) of the acceptor dye (CY3) and re-measuring fluorescence at each wavelength. If FRET was initially present, a resultant increase in donor fluorescence will occur upon photobleaching of the acceptor. The energy transfer efficiency was quantified as: FRET eff = (Dpost-Dpre)/Dpost, where Dpost is the fluorescence intensity of the donor (Alexa 488) after acceptor photobleaching, and Dpre the fluorescence intensity of the donor before acceptor photobleaching. An efficiency heatmap was generated for each cell. Following acquisition of the FRET images, an investigator blinded to treatment groups used the SMA image to select 10 regions of interest per cell that corresponded with SMA filaments. For each region of interest, FRET efficiency was calculated as above, and averaged to get a single value per cell.