Recombinant Anti-alpha smooth muscle Actin antibody [EPR5368] - BSA and Azide free (ab220795)

ab124964 staining alpha smooth muscle Actin in Mouse Uterus tissue sections by Immunohistochemistry (Formalin/PFA perfusion fixed frozen sections). Tissue samples were fixed by perfusion with formaldehyde, blocked with PB ab64226 for 10 minutes at Room temperature and antigen retrieval was by heat mediation in citrate buffer. The sample was incubated with primary antibody (1/2000) for 30 minutes. A HRP-conjugated Goat anti-rabbit polyclonal (undiluted) was used as the secondary antibody.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab124964).

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This data was developed using the same antibody clone in a different buffer formulation (ab124964).

Lanes 1 - 2: Merged signal (red and green). Green - ab124964 observed at 42 kDa. Red - loading control, ab7291 (Mouse anti-Alpha Tubulin [DM1A]) observed at 55kDa.

ab124964 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 ab124964 and ab7291 (Mouse anti-Alpha Tubulin [DM1A]) overnight at 4°C at a 1 in 10000 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.

Clone EPR5368 (ab220795) has been successfully conjugated by Abcam. This image was generated using Anti-alpha smooth muscle Actin antibody [EPR5368] (PE). Please refer to ab208844 for protocol details. 

Overlay histogram showing SV40LT-SMC cells stained with ab208844 (red line). The cells were fixed with 4% formaldehyde (10 min) and then permeabilized with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS / 10% normal goat serum to block non-specific protein-protein interactions followed by the antibody (ab208844, 1/5000 dilution) for 30 min at 22°C. 

Isotype control antibody (black line) was Rabbit IgG (monoclonal) Phycoerythrin (ab209478) used at the same concentration and conditions as the primary antibody. Unlabelled sample (blue line) was also used as a control. 

Acquisition of >5,000 events were collected using a 50 mW Yellow/Green laser (561nm) and 586/15 bandpass filter. 

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human stomach tissue labelling alpha smooth muscl Actin with purified ab124964 at 1/1000. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. A prediluted HRP-polymer conjugated anti-rabbit IgG was used as the secondary antibody. Negative control using PBS instead of primary antibody. Counterstained with Hematoxylin.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab124964).

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of normal human liver vessels tissue labelling alpha smooth muscle Actin with unpurified ab124964.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab124964).

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of normal human colon smooth muscle tissue labelling alpha smooth muscle Actin with unpurified ab124964.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab124964).

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human prostatic carcinoma smooth muscles tissue labelling alpha smooth muscle Actin with unpurified ab124964.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab124964).

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

Clone EPR5368 (ab220795) has been successfully conjugated by Abcam. This image was generated using Anti-alpha smooth muscle Actin antibody [EPR5368] (Alexa Fluor® 488). Please refer to ab202295 for protocol details.

ab202295 staining alpha smooth muscle Actin in SV40LT-SMC cells. The cells were fixed with 4% formaldehyde (10 min), 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 overnight at +4°C with ab at a 1/100 dilution (shown in green) and ab195889, Mouse monoclonal to alpha Tubulin (Alexa Fluor® 594), at a 1/250 dilution (shown in red). Nuclear DNA was labelled with DAPI (shown in blue).

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

This product also gave a positive signal under the same testing conditions in SV40LT-SMC cells fixed with 100% methanol (5 min)

Immunocytochemistry/Immunofluorescence analysis of HeLa cells labeling alpha smooth muscle Actin with ab220795 at 1/500. Cells were fixed with 4% paraformaldehyde and permeabilised with 0.1% tritonX-100. An Goat anti rabbit IgG (Alexa Fluor^® 488)
ab150077 (1/1000) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain.

Clone EPR5368 (ab220795) has been successfully conjugated by Abcam. This image was generated using Anti-alpha smooth muscle Actin antibody [EPR5368] (Alexa Fluor® 647). Please refer to ab202296 for protocol details.

ab202296 staining alpha smooth muscle Actin in SV40LT-SMC cells. The cells were fixed with 4% formaldehyde (10 min), 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 overnight at +4°C with ab202296 at 1/5000 dilution (shown in red) and ab195887, Mouse monoclonal to alpha Tubulin (Alexa Fluor^® 488), at 1/250 dilution (shown in green). Nuclear DNA was labelled with DAPI (shown in blue).

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

This product also gave a positive signal under the same testing conditions in SV40LT-SMC cells fixed with 100% methanol (5min).

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis showing vascular smooth muscle staining in skeletal muscle tissue using  alpha smooth muscle Actin with unpurified ab124964. Note positive staining on smooth muscle cells but negative on striated muscle cells.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab124964).

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human stomach carcinoma smooth muscles tissue labelling alpha smooth muscle Actin with unpurified ab124964.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab124964).

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of normal human tonsil vessels tissue labelling alpha smooth muscle Actin with unpurified ab124964.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab124964).

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

This IHC data was generated using the same anti-alpha smooth muscle Actin antibody clone, EPR5368, in a different buffer formulation (cat# ab124964).

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human ovary tissue labelling alpha smooth muscle Actin with unpurified ab124964 at 1/1000 dilution.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

This IHC data was generated using the same anti-alpha smooth muscle Actin antibody clone, EPR5368, in a different buffer formulation (cat# ab124964).

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human heart tissue labelling alpha smooth muscle actin with unpurified ab124964 at 1/1000 dilution. Note positive staining on smooth muscle cells but negative on striated muscle cells. 

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

Immunocytochemistry/Immunofluorescence analysis of A-673 (human muscle Ewing's Sarcoma cell line) cells labelling alpha smooth muscle Actin with purified ab124964 at 1/300. Cells were fixed with 4% paraformaldehyde. An Alexa Fluor^® 555-conjugated goat anti-rabbit IgG (1/500) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain.

Control: primary antibody (1/300) and secondary antibody, ab150113, an Alexa Fluor^® 488-conjugated goat anti-mouse IgG (1/500).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab124964).

Intracellular Flow Cytometry analysis of Jurkat (human T cell leukemia cell line from peripheral blood) cells labelling alpha smooth muscle Actin with purified ab124964 at 1/30 (red). Cells were fixed with 2% paraformaldehyde. A FITC-conjugated goat anti-rabbit IgG (1/150) was used as the secondary antibody. Green - Isotype control, rabbit monoclonal IgG. 

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab124964). 

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab124964).