IHC image of PAX5 staining in a section of formalin-fixed paraffin-embedded normal mouse spleen 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 20mins. The section was then incubated with ab211293, 0.73µg/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 hematoxylin 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.

 

IHC image of PAX5 staining in a section of formalin-fixed paraffin-embedded normal human TONSIL* 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 20mins. The section was then incubated with ab211293, 0.73 µg/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 hematoxylin 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.

All lanes : Anti-PAX5 antibody [1H9] (ab211293) at 1 µg/ml

Lane 1 : Human tonsil whole tissue lysate
Lane 2 : Ramos whole cell lysate
Lane 3 : Daudi whole cell lysate
Lane 4 : Mouse spleen whole tissue lysate
Lane 5 : Rat spleen whole tissue lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rat IgG H&L (HRP) preadsorbed (ab7097)

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 42 kDa
Observed band size: 45 kDa why is the actual band size different from the predicted?


Exposure time: 10 seconds

This blot was produced using a 4-20% Criterion TGX gel in 1X SDS buffer. The gel was run at 180V for 40 minutes before being transferred onto a Nitrocellulose membrane at 25V for 7 minutes using the Trans-Blot turbo transfer system. The membrane was then blocked for an hour using 3% milk before being incubated with ab211293 overnight at 4°C at 1µg/ml. Antibody binding was detected using a Goat anti-Rat antibody conjugated to HRP, and visualised using Bio-Rad Clarity Western ECL substrate.