When fixating bone to observe the expression of GFP from GFP expressing reporter mouse it inhibits staining for fluorescence microscopy. We can observe the GFP in PFA fixed bone samples but then the commercial antibodies available doesn't bind to those samples. Reducing the fixation timing to stain with other antibodies decreases the GFP expressing cell population.
What kind of antibodies are you using? I used bone marrow that I fixed I think with methyl based fixative, and then did a quick DAPI/ GFP antibody stain. I think this would work with other antibodies. I can find more details out about the protocol but you definitely don't fully go through IF/ IHC (no blocking etc). Basically you just fix the cells and do a quick stain. I don't see why costaining with another antibody wouldn't work. But, maybe it's harder in actual bone.
The antibodies against GFP that we use are JL8 from Clontech which is raised in mouse and also ab290 from abcam. Both are very robust against our Rosa25 driven Cre reporter.
Thank you for your response Teresa, but it seems you couldn't understand my problem. As I have written we have GFP expressing reporter mouse and in the bone sample we would like to see the GFP expression without using the antibody for it. I further want to stain the same bone sample with other antibodies and would like to see the co expression of GFP and the other antibody I am using. Problem is that in bone sample without fixing it with PFA (fixing preserves the endogenous GFP) you can't see the endogenous GFP expression but then it hampers with the binding capacity of other antibodies. That is why I was wondering if any one has an alternative way of doing this.
Have you tried cryosections (without fixation)? You do need a good tungsten steel knife to cut undecalcified bone, but I have had good results with many antibodies using this approach.
Hi Rob Van't Hof,
The reason we are trying to cryosection the bone after PFA fixation is that we want to preserve the endogenous GFP expression in those reporter mice bone sample and try for the staining with other antibodies . We did try to cryosection them without fixation but then the there was no GFP.
You could try one if the urea (Scale) based clearing methods to preserve GFP. http://www.nature.com/neuro/journal/v14/n11/full/nn.2928.html
It is possible to maintain GFP activity with short term fixation. There are acetone/PFA methods in the literature that maintain fluoresence. The acetone keeps the GFP protein (water soluble) from moving out of the tissue and into solution.
Another approach would be to simply use different fluorescent secondary antibodies for GFP and whatever other gene of interest. There are very specific GFP antibodies that give very little, if any, backround signal.
Good luck!
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