α smooth muscle actin antibody

The content of F-actin and G-actin in smooth muscle was measured using a method as previously described [21 (link), 22 (link)]. Briefly, tissues were treated with F-actin stabilization buffer (50 mM PIPES, pH 6.9, 50 mM NaCl, 5 mM MgCl₂, 5 mM EGTA, 5 % glycerol, 0.1 % Triton X-100, 0.1 % Nonidet P40, 0.1 % Tween 20, 0.1 % beta-mercapto-ethanol, 1 mM ATP, 1 μg/ml pepstatin, 1 μg/ml leupeptin, 10 μg/ml benzamidine). The supernatants of protein extracts were collected after centrifugation at 151,000 g for 60 min at 37 °C. The pellets were resuspended in ice-cold H₂O plus 1 μM cytochalasin D and then incubated on ice for 1 h to dissociate F-actin. The resuspended pellets were gently mixed every 15 min. The supernatant of the resuspended pellets was collected after centrifugation at 16,100 g for 2 min at 4 °C. Equal volume of the first supernatant (G-actin) or second supernatant (F-actin) was subjected to immunoblot analysis using α-smooth muscle actin antibody (Sigma). The amount of F-actin and G-actin was determined by scanning densitometry.

All animal experiments were performed according to Tel Aviv University ethical use protocols. Recipient SD male rats (200–240 g, Envigo Laboratories, Israel) were anesthetized using a combination of Ketamine (40 mg/kg) and Xylazine (10 mg/kg). Subcutaneous implantation of samples was performed by creating a small incision to the back and the scaffolds were inserted into the cavity. The electrodes were connected to a stimulus generator (STG-4002, Multichannel systems), and a voltage of 2 V was applied for 2 min once a day. Eight days after transplantation the rats were sacrificed and the samples were extracted, fixed in 4% formaldehyde, and embedded in optimal cutting temperature (OCT) compound. Using a cryotome™ FSE (Thermo scientific), 12 μm thick sections were prepared and affixed to X-tra® adhesive glass slides (Leica Biosystems, Wetzler, Germany). The fixed samples were washed three times in 1:10 PBS to extract the OCT compound. The samples were incubated with primary α-smooth muscle actin antibody (1:200, Sigma–Aldrich), washed three times and incubated for 1 h with Alexa Fluor 647 conjugated goat anti-mouse antibody (1:500; Jackson, West Grove, PA). For nuclei detection, the cells were incubated for 3 min with Hoechst 33,258 (1:1000; Sigma) and washed three times. Samples were visualized using a scanning laser confocal microscope (Nikon Eclipse Ni).