Application
Immunocytochemistry
Sample
African Green Monkey Cultured Cells (COS-7, kidney epithelium)
Specification
COS-7, kidney epithelium
Fixative
Paraformaldehyde
Permeabilization
Yes - Triton-X-100
Blocking step
BSA as blocking agent for 10 minute(s) · Concentration: 1% · Temperature: 20°C
Other product details
Dilution
1/100
Incubation time
1 hour(s) and 0 minute(s) · Temperature: 20°C · Diluent: PBS
Secondary antibody
Name
Non-Abcam antibody was used: Donkey-anti-Rabbit-TRITC, Jackson Immuno
Host species: Donkey
Clonality: Polyclonal
Conjugation: TRITC
Host species: Donkey
Clonality: Polyclonal
Conjugation: TRITC
Dilution
1/100
Additional data
Additional Notes
The antibody does not work with a standard fixation for ICC/IF as it does not recognize the non-denatured protein. However, applying a fixation for hidden antigens (modified from Peränen J, Rikkonen M, Kääriäinen L. A method for exposing hidden antigenic sites in paraformaldehyde-fixed cultured cells, applied to initially unreactive antibodies. J Histochem Cytochem. 1993 Mar41(3):447-54) results in very good recognition of VDAC/porin. Denaturing is achieved by Guanidine hydrochloride in 50 mM Tris-HCL pH 7.5 after fixation (3% pFA, 0.05% GA) and permeabilization (0.2% TX-100).
Abcam response
Thank you for your feedback and for your effort in making this antibody working. Your experience is a brilliant example that in science there is no perfect standard procedure which cannot be optimized.
Even ICC was performed with PFA as fixing agent, which usually does not require further treatment, but in this case an antigen retrieval step reviled the target and transformed a false negative into the correct results.
Even ICC was performed with PFA as fixing agent, which usually does not require further treatment, but in this case an antigen retrieval step reviled the target and transformed a false negative into the correct 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.
Dr. Michael Schrader
Verified customer
提交于 Feb 18 2010