IHC image of alpha smooth muscle Actin staining in a section of formalin-fixed paraffin-embedded normal human colon*, performed on a Leica BOND™. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20mins. The section was then incubated with ab203696, 1/100 dilution, for 15 mins at room temperature. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. The inset negative control image is taken from an identical assay without primary antibody.

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

All lanes : HRP Anti-alpha smooth muscle Actin antibody [1A4] (ab203696) at 1/5000 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


Exposure time: 20 seconds

ab203696 was shown to react with alpha smooth muscle Actin (HRP) in HeLa wild-type cells in western blot. Loss of signal was observed when ACTA2 knockout sample was used. Membranes were blocked in 3% milk in TBS-T (0.1% Tween^®) before incubation with ab203696 overnight at 4°C at a 1 in 5000 Dilution and ab184095 (Mouse Anti-GAPDH antibody [mAbcam 9484] - Alexa Fluor^® 680) at a 1 in 1000 dilution. Blots were developed with Optiblot ECL reagent (ab133456) and imaged.

All lanes : HRP Anti-alpha smooth muscle Actin antibody [1A4] (ab203696) at 1/5000 dilution

Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
Lane 2 : A431 (Human epithelial carcinoma cell line) Whole Cell Lysate
Lane 3 : Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate

Lysates/proteins at 10 µg per lane.

Developed using the ECL technique.

Performed under reducing conditions.

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


Exposure time: 1 minute

This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 2% Bovine Serum Albumin before being incubated with ab203696 overnight at 4°C. Antibody binding was visualised using ECL development solution ab133406.