>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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