Anti-alpha smooth muscle Actin antibody [1A4] (ab7817)

Ab7817 staining alpha smooth muscle actin in Mouse intestine tissue by Immunohistochemistry-Immunofluorescence. Tissue was fixed with formaldehyde and blocked with 100% Cas-block for 30 minutes at room temperature; antigen retrieval was performed by heat mediated citrate buffer, pH6. The sample was incubated with primary antibody at 1/200 dilution for 16 hours at 4°C. An Alexa Fluor^® 488 Goat anti-mouse IgG was used as the secondary antibody at 1/400 dilution. Autofluorescence was blocked with 0.1% Sudan Black in 70% ethanol for 10 minutes at room temperature after antigen retrieval, and followed with 3X wash with PBS-T after antigen retrieval. Image was taken with confocal microscope.

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IHC image of alpha smooth muscle actin staining in a human breast ductal carcinoma formalin fixed paraffin embedded tissue section*, performed on a Leica Bond™ system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab7817, 0.1µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

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ab7817 staining alpha smooth muscle Actin (green) in Mouse primary colon myofibroblasts by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with acetone and blocked with 5% BSA for 30 hours at 25°C. Samples were incubated with primary antibody (1/100 in PBS + 5% BSA) for 2 hours at 25°C. Donkey Anti-Mouse IgG H&L (DyLight® 488) (ab96875) (1/1000) was used as the secondary antibody. Costained with ab92547, Rabbit anti-Vimentin (red).

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ab7817 staining alpha smooth muscle Actin in human brain tissue sections by Immunohistochemistry (PFA perfusion fixed frozen sections). Tissue samples were fixed by perfusion with paraformaldehyde, cut into 20 micron slices, permeablized with 0.1 M PBS with 1% Triton X and blocked with 10% serum for 60 minutes at 24°C. The sample was incubated with primary antibody (1/200 in 0.1M PBST with 10% donkey serum) at 4°C for 24 hours. An Alexa Fluor^® 568-conjugated donkey monoclonal (1/1000) was used as the secondary antibody.

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ab7817 stained in SV40LT-SMC cells. The cells were fixed with 4% paraformaldehyde (10min) at room temperature and incubated with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% triton for 1h at room temperature to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab7817 at 5µg/ml overnight at +4°C. The secondary antibody was Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) preadsorbed (ab150117) (colored green) used at 1 ug/ml for 1hour at room temperature. DAPI was used to stain the cell nuclei (colored blue) at a concentration of 1.43µM for 1hour at room temperature.

Davis FM et al investigates the differentiation potential of mammary stem cells in adults. Ab7817 staining smooth muscle actin in EYFP+ cells from R26^[CA]30EYFP mouse at 1:200 dilution. 

ab7817 staining alpha smooth muscle Actin in human IMR-90 (Human Lung Fibroblast Cell Line) cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with paraformaldehyde, permeabilized with 0.1% TritonX-100 and blocked with 100% Cad-Block for 30 minutes at room temperature. Samples were incubated with primary antibody 1/200 in antibody diluent buffer for 16 hours at 4°C. An Alexa Fluor^® 488-conjugated polyclonal Goat anti-mouse IgG, dilution 1/400, was used as secondary antibody.

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ab7817 staining alpha smooth muscle actin in Human liver tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with paraformaldehyde and permeabilized with wash buffer with tween; antigen retrieval was by heat mediation in Tris-EDTA buffer, pH 9.0. Samples were incubated with primary antibody (1/100 in blocking buffer) for 30 minutes at 20°C. A HRP-conjugated Goat anti-mouse IgG polyclonal (undiluted) was used as the secondary antibody.

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Overlay histogram showing SV40LT-SMC cells stained with ab7817 (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 (ab7817, 0.1µg/1x10⁶ cells) for 30 min at 22°C. The secondary antibody used was Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) (ab150113) at 1/2000 dilution for 30 min at 22°C

Isotype control antibody (black line) was mouse IgG2a [18C8BC7AD10] (ab170191) 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 Blue laser (488nm) and 530/30 bandpass filter.

This antibody gave a positive signal in HeLa cells fixed with 80% methanol (5 min)/permeabilized with 0.1% PBS-Triton X-100 for 15 min used under the same conditions.

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