Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Rat colon tissue sections labeling alpha smooth muscle Actin with ab150301 at 1/200 dilution (0.26 µg/ml). Heat mediated antigen retrieval was performed Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 10 mins. Goat Anti-Rabbit & Mouse IgG (HRP) was used as the secondary antibody. Negative control: PBS instead of the primary antibody. Hematoxylin was used as a counterstain.
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab150301)

Immunocytochemistry/ Immunofluorescence analysis of NIH/3T3 (mouse embryonic fibroblast) cells labeling alpha smooth muscle Actin with purified ab150301 at 1/100(1.65 µg/ml). 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) 1/200 (2.5 µg/ml). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) was used as the secondary antibody at 1/1000 (2 µg/ml) dilution. DAPI (blue) was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab150301).

All lanes : Anti-alpha smooth muscle Actin antibody [SP171] (ab150301) at 1/130 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : ACTA2 knockout HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 42 kDa
Observed band size: 42 kDa

This data was developed using the same antibody clone in a different buffer formulation (ab150301).

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.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Mouse colon tissue sections labeling alpha smooth muscle Actin with ab150301 at 1/200 dilution (0.26 µg/ml). Heat mediated antigen retrieval was performed Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 10 mins. Goat Anti-Rabbit & Mouse IgG (HRP) was used as the secondary antibody. Negative control: PBS instead of the primary antibody. Hematoxylin was used as a counterstain.
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab150301)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human colon tissue sections labeling alpha smooth muscle Actin with ab150301 at 1/200 dilution (0.26 µg/ml). Heat mediated antigen retrieval was performed Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 10 mins. Goat Anti-Rabbit & Mouse IgG (HRP) was used as the secondary antibody. Negative control: PBS instead of the primary antibody. Hematoxylin was used as a counterstain.
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab150301)

Immunohistochemical analysis of formalin fixed, paraffin embedded human colon tissue labeling alpha smooth muscle Actin with ab150301 at 1/200 dilution.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA and sodium azide (ab150301)

This data was developed using the same antibody clone in a different buffer formulation (ab150301). ab150301 staining alpha smooth muscle Actin in wild-type HeLa cells (top panel) and ACTA2 knockout HeLa cells (bottom panel). The cells were fixed with 100% methanol (5 min) then permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab150301 at 5μg/ml concentration and ab7291 (Mouse monoclonal to alpha Tubulin) at 1/1000 dilution overnight at 4°C followed by a further incubation at room temperature for 1h with a goat secondary antibody to rabbit IgG (Alexa Fluor^® 488) (ab150081) at 2 μg/ml (shown in green) and a goat secondary antibody to mouse IgG (Alexa Fluor^® 594) (ab150120) at 2 μg/ml (shown in red). Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems TCS SP8).

Flow Cytometry analysis of HeLa (human cervix adenocarcinoma epithelial cell) 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.
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab150301).

Flow Cytometry analysis of C6 (Rat glial tumor glial cell) 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.
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab150301).

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.
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab150301).