Recombinant Anti-alpha smooth muscle Actin antibody [SP171] (ab150301)

Fluorescence multiplex immunohistochemical analysis of human liver tissue (formalin-fixed paraffin-embedded section). Panel A shows merged staining of ab195872 anti-Cytokeratin 19 stained on branch of bile ducts (magenta; Opal™690) at 1:8000 ( 0.127 μg/ml) [Panel B], ab275376 anti-Factor VIII stained on endothelial cells (red; Opal™570) at 1:1000 ( 0.457 μg/ml) [Panel C], and ab150301 anti-alpha smooth muscle Actin stained on smooth muscles (green; Opal™520) at 1:200 ( 0.14 μg/ml) [Panel C] on human liver. DAPI was used as a nuclear counter stain. Followed by Opal Polymer HRP Ms + Rb secondary. The immunostaining was performed on a Leica Biosystems BOND® RX instrument with an Opal™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope. The section was incubated in three rounds of staining: in the order of ab195872 for 30 mins, ab275376 for 30 mins and ab150301 for 10 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins was used. 

Lanes 1 - 2: Merged signal (red and green). Green - ab150301 observed at 42 kDa. Red - loading control, ab7291 (Mouse anti-Alpha Tubulin [DM1A]) observed at 55kDa.

ab150301 was shown to react with alpha smooth muscle Actin in wild-type HeLa cells in western blot. Loss of signal was observed when ACTA2 knockout sample was used. Wild-type HeLa and ACTA2 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween^®) before incubation with ab150301 and ab7291 (Mouse anti-Alpha Tubulin [DM1A]) overnight at 4°C at a 1 in 130 Dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

Immunocytochemistry/ Immunofluorescence analysis of NIH/3T3 (mouse embryonic fibroblast) cells labeling alpha smooth muscle Actin with purified ab150301. Cells were fixed in 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Cells were counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) was used as the secondary antibody. DAPI (blue) was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

Flow Cytometry analysis of HeLa (human cervix adenocarcinoma epithelial cell) cells labeling alpha smooth muscle Actin with purified ab150301 (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) was used as the secondary antibody. Isotype control - Rabbit monoclonal IgG (ab172730) / Black. Unlabeled control - Unlabelled cells / blue.

Flow Cytometry analysis of C6 (Rat glial tumor glial cell) cells labeling alpha smooth muscle Actin with purified ab150301 (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) secondary antibody was used. Isotype control - Rabbit monoclonal IgG (ab172730) / Black. Unlabeled control - Unlabelled cells / blue.

Flow Cytometry analysis of NIH/3T3 (Mouse embryonic fibroblast) cells labeling alpha smooth muscle Actin with purified ab150301 at 1/200 dilution (0.825 µg/ml) (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) secondary antibody was used at 1/2000 dilution. Isotype control - Rabbit monoclonal IgG (ab172730) / Black. Unlabeled control - Unlabelled cells / blue.