Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Mouse Tissue sections (mammary tumor)
Specification
mammary tumor
Fixative
Paraformaldehyde
Antigen retrieval step
Heat mediated
Permeabilization
No
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C
Other product details
Dilution
1/100
Incubation time
1 hour(s) and 0 minute(s) · Temperature: 22°C · Diluent: DAKO Background Reducing Diluent
Secondary antibody
Name
Non-Abcam antibody was used: Sigma anti-Chicken HRP
Host species: Rabbit
Clonality: Polyclonal
Conjugation: Horse Radish Peroxidase
Host species: Rabbit
Clonality: Polyclonal
Conjugation: Horse Radish Peroxidase
Dilution
1/1000
Additional data
Additional Notes
I have only tried this antibody one time, but it gave a TON of background. I may try to troubleshoot and use again, but it didn't work as I had hoped.
Abcam response
We recommended the following changes:
1) Test both shorter and longer antigen retrieval times as well as a different buffer e.g. Tris-EDTA pH 9 instead of citrate buffer.
2) Increase blocking e.g. 10% serum for 1hr room temperature.
3) Reduce antibody, both primary and secondary. Perform a secondary
antibody control (no primary antibody) to determine whether the background is
caused by the primary or secondary antibody.
The user has not replied so we do not know if these recommendations improved his/her results.
1) Test both shorter and longer antigen retrieval times as well as a different buffer e.g. Tris-EDTA pH 9 instead of citrate buffer.
2) Increase blocking e.g. 10% serum for 1hr room temperature.
3) Reduce antibody, both primary and secondary. Perform a secondary
antibody control (no primary antibody) to determine whether the background is
caused by the primary or secondary antibody.
The user has not replied so we do not know if these recommendations improved his/her results.
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.
Abcam user community
Verified customer
投稿 Aug 28 2007