This data was developed using the same antibody clone in a different buffer formulation that is PBS and sodium azide free (ab240654)

ab240654 staining alpha smooth muscle Actin in SV40LT-SMC cells. The cells were fixed with 4% paraformaldehyde (10 min), permeabilized with 0.1% PBS-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 ab240654 at 1µg/ml and ab6046, Rabbit polyclonal to beta Tubulin - Loading Control. Cells were then incubated with ab150117, Goat polyclonal Secondary Antibody to Mouse IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 dilution (shown in green) and ab150080, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 594) at 1/1000 dilution (shown in pseudocolour red). Nuclear DNA was labelled with DAPI (shown in blue).

Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

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, 0.034µg/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

Lanes 1 - 2: Merged signal (red and green). Green - ab7817 observed at 42 kDa. Red - loading control, ab52866 (Rabbit anti-alpha Tubulin antibody [EP1332Y]) observed at 55kDa.

ab7817 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 ab7817 and ab52866 (Rabbit anti-alpha Tubulin antibody [EP1332Y]) overnight at 4°C at a 1 in 131.58 Dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Mouse IgG H&L (IRDye^® 800CW) preabsorbed (ab216772) and Goat anti-Rabbit IgG H&L (IRDye^® 680RD) preabsorbed (ab216777) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

Overlay histogram showingSV40LT-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, 0137µg/ml) for 30 min at 22°C. The secondary antibody used wasGoat 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.

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 0.034µg/ml 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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Formalin-fixed, paraffin-embedded Pig kidney tissue stained for alpha smooth muscle Actin (Green) using ab7817 at 0.034µg/ml followed by aDonkey anti-Mouse IgG (H+L) Alexa Fluor® 647 secondary antibody at 1/300 dilution.

Antigen retrieval:Heat mediated - Buffer/Enzyme Used: Citrate Buffer pH 6.0

The alpha smooth muscle Actin antibody signal is pseudo colored Green. The nuclei are Blue using Hoechst stain. The background tissue is stained Red

ab7817 stainingalpha smooth muscle actininHumanliver tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with paraformaldehyde andpermeabilized with wash buffer with tween; antigen retrieval was by heat mediation inTris-EDTA buffer, pH 9.0. Samples were incubated with primary antibody (0.034µg/ml in blocking buffer) for30 minutesat 20°C. A HRP-conjugatedGoat anti-mouse IgG polyclonal (undiluted) was used as the secondary antibody.

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Immunohistochemical analysis of mouse aorta (A) or skin (B) tissue, staining alpha smooth muscle Actin with ab7817.Tissue was fixed with 10% Neutral Buffered Formalin and blocked with 1% serum for 45 minutes 21°C; antigen retrieval was by enzymatic method in 0.0001% Trypsin-CaCl. Samples were incubated with primary antibody (0.034µg/ml in 0.3% Triton X-100 in PBS) for 1 hour at 21&degC. A biotin-conjugated horse anti-mouse polyclonal IgG (1/50) was used as the secondary antibody.

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ab7817 stainingalpha smooth muscle Actin(green)inMouse primary colon myofibroblastsby ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed withacetone and blocked with 5% BSA for30hours at 25°C. Samples were incubated with primary antibody (1/100 in PBS + 5% BSA) for2 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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Davis FMet 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 0.034µg/ml.

ab7817 staining alpha smooth muscle Actin inhumanIMR-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 3.41µg/ml in antibody diluent buffer for 16hours 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 mouse heart cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with paraformaldehyde, permeabilized with TritonX-100 and blocked with 5% BSA for 30 minutes at room temperature. Samples were incubated with primary antibody 6.82µg/ml in blocking buffer for 2 hours. An Alexa Fluor^® 488-conjugated Donkey monoclonal to mouse IgG, dilution 1/200, was used as secondary antibody.

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