[Histonet] GFP labelled cells after decalcification...are they still labelled?

[Adam .]

Hi James,

I do this all the time in mouse bones. I've found that EGFP is very robust,
and it can survive fixation with PFA and Zn buffered formalin, as well as
decalcification with EDTA or formic acid, followed by paraffin embedding.
However, the endogenous fluorescence is highly quenched by this process and
barely detectable over background if at all. In order to detect it post
embedding, I use the chicken anti-GFP polyclonal from Abcam, and a
fluorophore conjugated donkey anti-chicken IgY with very good results. If
you want endogenous fluorescence, you can fix the bones in 4% PFA and
decalcify using EDTA, and use frozen sections.

I hope this helps,
Adam

On Thu, Mar 31, 2011 at 12:42 PM, James Hart <jameshart77 <@t> gmail.com> wrote:

> Hi All
>
> A first time user of Histonet here -with a quick question for research
> histologists...
>
> Has anyone ever attempted to locate GFP expressing cells in
> bone-marrow sections after decalcification and paraffin embedding? (I guess
> my question really is: Does the decalcification process destroy or weaken
> the GFP signal)
>
> Any advice would be greatly appreciated!
>
> Regards
>
> James
>
> Dr James Hart BVSc MS
> Comparative Orthopaedics, Cornell University
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