Re: [Histonet] GFP and decalcification

From:"Andrea Hooper"

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 
>this problem. 
>Good luck
>Gayle M. Callis
>Bozeman, MT
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