Immunocytochemistry/ Immunofluorescence abreview for Anti-HEF1 / NEDD-9 antibody [2G9]

Inconclusive
Abreviews
Application
Immunocytochemistry/ Immunofluorescence
Sample
Mouse Cell (embryonic stem)
Permeabilization
Yes - 0.5% triton-x-100 in PBS
Specification
embryonic stem
Blocking step
BSA as blocking agent for 30 minute(s) · Concentration: 3% · Temperature: 25°C
Fixative
Paraformaldehyde

Other product details

Incubation time
1 hour(s) and 0 minute(s) · Temperature: 25°C · Diluent: 3% BSA in PBS with 0.1% TX-100
Dilution
1/500

Secondary antibody

Dilution
1/400
Name
Non-Abcam antibody was used: donkey anti-mouse 555
Host species: Donkey
Clonality: Polyclonal
Conjugation: Alexa Fluor® 555

Additional data

Additional Notes
Not sure if the observed pattern is real HEF1 staining or background HEF1 is supposed to localize to focal adhesions but don't see much co-localization with actin here. I also got the same results with a 4% PFA followed by a 100% methanol fix. Image shows mouse embryonic stem cells stained for DNA (DAPI, blue), actin (phalloidin, green) and HEF1 (red).
Abcam response
We have had no previous complaints for this antibody. We have no data on the PFA fixation length and permeabilization step from the customer. Protocol tips were given to the customer: 1. Incubate primary antibody overnight 2. Block with donkey serum The above should help obtain specific stronger staining.

Prof. Reiter Lab

Verified customer

Submitted Jul 07 2010

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