I am trying to preform an immunohistofluorescence in GFAP-eGFP transgenic mouse cortical brain sections. The protocol involves antigen retrieval with citrate buffer followd with blocking, washing and incubation with primary and secondary antibodies. The experiment went fine for all the antibodies I worked with but always I lose the expression of eGFP from the transgenic mouse. I'll appreciate if someone can guide me in this regard.
Hi Ambey Shree Shukla, you can try to amplificate your signal using an anti-eGFP primary antibody.
Hi Ambey,
First try what Martina recommended above.
However, in the past I had the problem that the antigen retrieval disrupted so much the folding of the GFP that it won't be recognized by the polyclonal antibodies I used. If direct amplification doesn't work, try the following:
Perform the complete staining for GFP on your samples (e.g. Permeabilization, without blocking primary antiGFP+secondary[488]), wash like you usually do and fix everything again with PFS 2%(*) for 10-15 minutes. Wash the PFA, let slices dry a bit so they re-attach to the slide and proceed with the antigen retrieval as usual and when finished check to confirm the amplified signal was retained. Afterwards stain for your other antibodies as usual. Alexa fluorophores are incredibly thermostable, and will hold on at the 90-95 C of the antigen retrieval. I successfully tried 95C over an hour. This protocol typically works like a charm for epitopes that are lost or destroyed with antigen retrieval.
(*) Increasing the PFA% to 4% will increase the retention of the signal but may affect the staining of other epitopes. Using PFA 2% is a good compromise.
Hope it works for you as good as it does for me.
Good luck!
Hi Ambey, when you are woking with frozen sections and Anti-GFAP, actually it is not necessary to work with antigene retrieval. Most the time this working steps are recommanded for formaline fixed paraffin embedded (FFPE) tissue.
So what I would do is what Martina tells you about anti-GFP. You can prepare a antibody cocktail when Anti-GFP and your other antibody of interest are generated in different host (e.g. mouse and rabbit). to find out which antibody consentration and additional treatment you need, run different concentrations and or detection systems (flu or biotin labeled secondary antibody or Streptavidin labelled with fluorophores). if you need a antigene retrieval us this enhancement in the second step, ohterwise you would enhance all eptopes evan those which not need to beenhanced. Good luck!
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