Hi there, I'm hoping someone may be able to help me. I have been trying (and failing) to conduct IHC staining on liver tissues collected from a cre+ mouse line which should be expressing GFP following the introduction of tamoxifen. The mice have been genotyped and are GFP positive, the tissue was collected 7 days post tamoxifen-induced cre recombination and the tissues were fixed with 10% formalin overnight. A positive control tissue sample from another mouse line with identical tamoxifen dosing and tissue fixation was used in which membrane GFP was successfully detected by DAB staining (heat mediated antigen retrieval in Tris-EDTA, primary antibody: ab13970). I have tried 2 GFP primary antibodies thus far (ab13970 and ab183734) but because the positive control was a success I don't think the antibody is the issue.
Can someone please direct me towards a tried and tested method for detecting nuclear GFP expression? I'm assuming there may be some permeabilization steps that I'm missing or potentially having fixed the tissues in formalin rather than cryofixing or fixing with 4% PFA may be an issue? Any recommendations will be extremely appreciated!
Hi Caitlin, to compare positive control and your experimental tissue you have to treate both samples in the same way. Same fixation protocol, same immuno protocol same antibodies. If there is any difference between pos. control and exp. tissue then it could be the answer of your question why the immuno protocol does not work on your liver tissue. You can try to find the positive signal in the fluorescence microscope. If the tamoxifen-application was working properly I think you could see somthing in the fluorescence microscope. If not, either the signal is too week or the tamoxifen-application didn't work. For fixed frozen tissue I would recommand ThermiFisher GFP-antibody A21311 without any antigen retrieval.
Hi Ute Neubacher thank you so much for your response, I apologise for taking so long to respond.
The positive control was treated identically to the other tissues - the only difference between them is when the GFP is localised following cre recombination - I do not, unfortunately, have access to a positive control tissue with GFP localised in the nucleus. I have ordered the A21311 antibody as per your suggestion - would it be possible for you to share the protocol that you follow when staining without an antigen retrieval step? Can you perform the antigen retrieval-less staining on tissues fixed in PFA/formalin or should I use a different method of fixation? Do you need to first permeabilize the tissue with triton X-100 washes?
Thank you so much for your time.
Hello Caitlin, I used PFA fixed in 30% sucrose cryo- protected 30µm thick brain sections. I treat them in the free floating way. That means you do the GFP-immuno before you mount the sections on slides. The protocol:
1. Blockin-step: 10% normal serum in 0,2% Triton-X in PBS (PBS-Tx) - 90 min;
2. Primary AB: rb-Anti-GFP 1:500 - 1:1000 in 1% normal serum PBS-Tx - overnight.
3. 3 x 10 min rinsing in PBS
4. Secondary-AB: Anti-rabbit -Fluophore (CY2,CY3,CY5) 1:250 - 1:500 in 1% normal serum in PBS-Tx - 90 min
5. 3 x 10 min rinsing in PBS
The reaction was carried out on rocking table at room temperatur (+/- 22°C)
6. mounting sections on gelatine coated slides, air drying in the fridge;
7. mounting with a mounting-medium containing Dapi.
Good luck!
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