We have mice expressing a Flag-tagged protein (only one flag epitope) at endogenous levels. We have major problems (non specific labeling; particularly strong in the olfactory bulb) with most antibodies in our perfusion-fixed brain sections. Anyone has ever used anti-FLAG antibodies to label mouse brain proteins? Any advice? Best fixative to be used? Aware of a specfiic antibody?
The following are issues to evaluate and pre-test when using IH biomarkers in brain tissue: the source and specifications of the antibody to be used. It is advisable to test several antibodies from several manufactures and read carefully the manufactures information and advice for use. Since your tissue is fixed, yes the fixative can block the reactivity of the epitome and the background staining could be linked at least in part to the fixative used. The manufactures usually provide info on this regard and if not I suggest to call their technical department. The may be able to help. Proper washing of the tissue after fixation and through staining procedure may also help to reduce background. There are several solutions that can be used including PBS all depends of the protocol you use. Antigen recovering procedures to enhance the antibody binding. There are several techniques e.g. microwave antigen recovering. These are general tips and considerations regardless the biomarker used.
I just read article about fixatives that might be of interest to you:
http://www.ncbi.nlm.nih.gov/pubmed/20026672
I found brain to be tricky for immunofluorescence. Could you maybe write more about how you are fixing and sectioning the tissue, as it may affect the background? Also is the unspecific staining originating from primary or secondary antibody (have you done only secondary ab control?)?
We did many controls thus far.
Fixative: Mice were perfusion-fixed with 4% PFA +/- 0.18% picric acid followed by 1h postfixation. We did perform control experiments without 1st Ab and we have no sugnal at all. Importantly, we did both fluorescence and DAB labeling and have the same non specific staining. I should mention here that the non specific staining is not like a background signal. It stains very specific cells or brain structures, with adjacent cells/structures aren't labelled.
What you describe looks like the primary antibody is crossreacting with a similar antigen. In this case you may try two things:
1. Test another brand of antibodies untill you find a proper one. But I think you wrote you have done that already a few times
2. Try to stain your samples with an antibody that you used once on a WT tissue. If the problem is not the recognition of an antigen similiar to the FLAG tag, but presence of some unspecifically binding antibodies, this step might help.
You might also play with the dilution of antibody to titrate it so it binds perferentially to the tag and not anything else. This should work if your antibody has more affinity to specific target than to unspecific.
Btw. I am now trying to optimize the perfusion/cryoembedding protocol and I am curious if you had done fixation with 4% PFA only? Is the addition of picric acid helping?
Thanks for your advise, we will continue to work it out.
As for the perfusion, we found that this is very specific to antigen and antibodies. In this particular case, picric acid didn't helped at all. But we have used other Abs for which adding picric acid was fantastic (see our J Neurosci paper and the NK1 staining).
As discussed with one of your student today. Despite the blast made with your flag in the olfactory bulb, there still appearance of non-specificity. Maybe to testing with blocking solution more concentrated. Currently, I understand that you work with the 3% but often with my olfactory bulbs blocking solution is between 5 and 10% depending on the case.
Hello Louis,
could you solve this? how? I am experiencing the same. Any suggestion will be appreciated.
Dear Ivan,
no, we have never been able to solve this problem. Sorry I cannot help at the moment.
Dear Louis, I have the same problem with anti-Flag Ab. Did you try different dilutions? Could it help?
We have tried many things so far, including Ab dilutions and different fixatives. We are able to IP the receptor using the Flag Ab, but we have not been able to label it using IHC thus far.
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