[Histonet] LacZ and decalcification

From:Nicole Collette

>Hello, All,
>There seems to be a lot of mystery surrounding this issue, I was 
>hoping someone might have a definitive answer. I have done lots and 
>lots of lacZ stains in mice- embryos, adults, all kinds of tissues. 
>I have followed published protocols that should never have been 
>published, and have recovered somehow to turn out some great lacZ 
>I have never had samples that survived automatic tissue processing 
>for paraffin, they end up completely unusable with artefactual lacZ 
>stain. I have been told that I can process as usual for paraffin 
>embedding (for tissues that don't need decalcification) as long as I 
>hand-process the tissue to limit the heat on the tissues. Is this 
>true? Or was I just doing the automatic processing all wrong??
>I have done frozen sections, post-stain, which turn out fine, but 
>only  up to E17.5 mouse embryos. I have done stain on slides after 
>sectioning on a few sections of adult bone, undecalcified, using the 
>CryoJane system, also with the stain working, but it has its 
>limitations, in that I'd like thicker sections to take better 
>photos, but they don't stick to the slides very well, since only 
>very thin works, even with the extra adhesive slides for hard 
>tissue. Thus, I am back at the decalcification issue.
>Can I decalcify with EDTA before stain? How long for adult mouse 
>bones, and what percentage of EDTA? I assume the pH is the most 
>important part of this issue, but I am worried about prolonged 
>decalcification with the limited fixation allowed for LacZ staining 
>to avoid osteoclast staining artefacts, and/or overfixation to 
>survive the long EDTA process (Will it kill the stain? or ruin the 
>tissue?). I would think the best way would be to stain the bones as 
>whole-mount tissues, then decalcify (how long and at what 
>percentage?) after post-fixation, at which point I have fewer 
>worries. Should I be concerned about stain penetration through 
>intact bone during stain as whole-mount (dissected free of adherent 
>tissue, of course).
>Any advice or wisdom would be most helpful.
>Thanks so much!

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