The decalcifier you used for two days is rather strong (8% nitric acid and 70% alcohol [made from 92%?]) The alcohol is added to the acid in order to slow down the effects of the acid on tissues but the nitric acid may have really damaged the GFP. Then you processed and embedded in paraffin?
You could try EDTA, using 10 to 14% tetrasodium EDTA (this will have a beginning alkaline pH) with pH adjusted down to pH 7 to 7.4 using glacial acetic acid. THis is much slower, but also less damaging. You might be lucky and retain GFP fluorescence. You should do endpoint testing to know when bone is decalcified. There is a clever weight loss/weight gain endpoint test originally used to test nitric acid decalcification, but works nicely with EDTA. I will be happy to send that as an attachment via private email. If you have an xray unit or microCT scanner, you can check endpoint with these units.
GFP is also sensitive to alcohols and heat, along with pH, so you may have damaged the GFP not only with the strong mineral acid decalcifier but also the processing reagents. The GFP is still there, but doesn't fluoresce anymore.
If you try EDTA decalcification, I suggest you do an Rabbit antiGFP followed by an antiRabbit secondary labelled with Alexa 488 or FITC. This way you locate the GFP site and match its fluorescent signal with a matching color (green like the GFP)
If your bone has been fixed in neutral buffered formalin, you will probably experience aldehyde induced autofluorescence which makes viewing the FITC signal a bit more difficult unless you can eliminate the autofluorescence chemically or by settings on a confocal or spectral imaging setup. Go to the IHCworld website - there is a superb discussion of autofluorescence and how to reduce this problem.
Gayle M. Callis
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