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I have a reply from the customer. Regarding the IHC testing, I was under the impression that they used paraffin sections, however, they used sections that were slightly fixed, impregnated with Sucrose and frozen to cut on a freezing microtome. Can you please take a look at the IHC form again with this information in mind? Regarding the suggestions, they do not have a skin biopsy available for a positive control. They did buy the antibody for IHC and only tried it in WB after it did not work in IHC, so they do not want to try the WB again if they do not have to.
Do you have suggestions for the antibody in IHC?
Asked on Feb 02 2012
Thank you for your email.
I have a couple recommendation for your customer when using IHC-Fr.
1.) I strongly suggest to run a positive control. ICAM1 belongs to the homing receptors and to my knowledge should not be expressed in brain. These homing receptor are expressed in site with inflammation to accumulate lymphocytes. If treated skin biopsies are not available, lung or bone marrow might be suitable.
2.) According to the protocol your customer send us, the mouse brain is fixed twice: perfusion fixed and fixed for 4days after. This strong (double-)fixation can lead to confirmation changes in the target protein which renders the antibody unable to recognize the target protein.
I recommend to try various antigen retrieval methods. This has to be tested experimentally.
3.) Your customer is correct, this antibody is labeled with biotin. Without using a secondary antibody there is less amplification of the signal and I therefore suggest to dilute the antibody less and to try 1:50.
I hope these tips can help improve the result. Please do not hesitate to contact me again with any further questions.
Answered on Feb 02 2012