We use 10% EDTA, pH 6.95 on PFA perfused and then submersion fixed
murine bones and get nice GFP signal.
>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
>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
>Gayle M. Callis
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