IHC image of Elastin staining in a section of formalin-fixed paraffin-embedded normal human normal duodenum* performed on a Leica BOND^TM 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 20mins. The section was then incubated with ab9519, 1ug/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. The inset secondary-only 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

IHC image of ab9519 staining in 10% formaldehyde fixed frozen tissue section of normal human skin.

Non-specific protein-protein interactions were blocked using TBS containing 0.025% (v/v) Triton X-100, 0.3M (w/v) glycine and 3% (w/v) BSA for 1h at room temperature. The section was then incubated with ab9519 (1μg/ml) in TBS containing 0.025% (v/v) Triton X-100 and 3% (w/v) BSA overnight at +4°C. The section was then incubated with ab150119 (Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor® 647)) and DAPI for 1 hour at room temperature.

The DAPI only control (no antibody) inset shows no autofluorescence, demonstrating that any Alexa Fluor^® 647 signal is derived directly from bound ab9519. 

For other IHC staining systems (automated and non-automated), customers should optimize variable parameters such as antibody concentrations and incubation times.

ab9519 staining pig small intestine tissue sections by IHC-P.  Sections were fixed with Bouins and subjected to an enzymatic antigen retreival step.  The primary antibody was diluted 1/200 in a commercially available antibody diluent (replacing the blocking step) and incubated with the sample for 1 hour at 20°C.  A HRP conjugated goat anti-mouse antibody was used as the secondary.

See Abreview

Anti-Elastin antibody [BA-4] (ab9519) at 0.51 µg/ml + Human skin tissue lysate - total protein (ab30166) at 20 µg

Secondary
Goat Anti-Mouse IgG H&L (HRP) preadsorbed (ab97040) at 1/5000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 68 kDa
Observed band size: 70 kDa why is the actual band size different from the predicted?
Additional bands at: 55 kDa. We are unsure as to the identity of these extra bands.


Exposure time: 20 minutes