ab70475 stained MCF7 cells. The cells were 4% formaldehyde fixed for 10 minutes and then incubated in 1%BSA / 10% normal Goat serum / 0.3M glycine in 0.1% PBS-Tween for 1hour at room temperature to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab70475 at 10µg/ml) overnight at +4°C. The secondary antibody (pseudo-colored green) was ab150117 Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) preadsorbed used at a 1/1000 dilution for 1hour at room temperature. ab195889 Anti-alpha Tubulin (Alexa Fluor® 594) was used as a counterstaining (pseudo-colored red) at a 1/250 dilution for 1hour at room temperature. DAPI was used to stain the cell nuclei (pseudo-colored blue) at a concentration of 1.43µM for 1hour at room temperature.

This data was developed using the same antibody clone in a different buffer formulation containing PBS and sodium azide (ab70475).

IHC image of MUC1 staining in human normal colon 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 ab70475, 5µg/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

This data was developed using the same antibody clone in a different buffer formulation containing PBS and sodium azide (ab70475).

Ab70475 staining human normal lung. Staining is localised to the apical membrane.
Left panel: with primary antibody at 1 ug/ml. Right panel: isotype control.
Sections were stained using an automated system DAKO Autostainer Plus , at room temperature. Sections were rehydrated and antigen retrieved with the Dako 3-in-1 antigen retrieval buffers EDTA pH 9.0 in a DAKO PT Link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 minutes. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 minutes and detected with Dako Envision Flex amplification kit for 30 minutes. Colorimetric detection was completed with diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that for manual staining we recommend to optimize the primary antibody concentration and incubation time (overnight incubation), and amplification may be required.

This data was developed using the same antibody clone in a different buffer formulation containing PBS and sodium azide (ab70475).

All lanes : Anti-MUC1 antibody [HMFG1 (aka 1.10.F3)] (ab70475) at 1 µg/ml

Lane 1 : ab30090, 10 ug
Lane 2 : ab30051, 10 ug
Lane 3 : MCF7 (Human breast adenocarcinoma cell line) Whole Cell Lysate, 10 ug

Secondary
All lanes : Goat polyclonal to Mouse IgG - H&L - Pre-Adsorbed (HRP), at 1/5000 dilution

Performed under reducing conditions.

Predicted band size: 122 kDa


Exposure time: 4 minutes

This data was developed using the same antibody clone in a different buffer formulation containing PBS and sodium azide (ab70475).