[Histonet] Lineage tracing in bone

[Adam .]

Hi all,

As my thesis project moves forward, my commitee has asked me to do some
lineage tracing in mouse bone. For those of you not familiar with this, you
mate a mouse expressing a Cre recombinase to another mouse expressing a
reporter gene with a premature stop codon that can be floxed out. The
reporter is usually lacZ or GFP. The Cre will remove the stop codon and
allow expression of your reporter in the cells the cre is expressed in (and
all its progeny).

Here's the problem: I don't have access to a tape transfer system (yet) so I
have to use fixed decalcified bones. I would prefer to use paraffin embedded
sections due to the improved morphology. To make things more complicated. I
probably will even have to do colocalization with other antibodies.

So here are the options that I am familiar with for reporter strains:

1) lacZ reporters (http://jaxmice.jax.org/strain/003474.html)
I worry that the fixation or embedding will destroy the lacZ. It's pretty
common in developmental biology to see people use these for unfixed tissue
to directly assay lacZ. There was some discussion in the archives (
http://lists.utsouthwestern.edu/mailman/htdig/histonet/2008-February/035626.html)
on a method to embed in paraffin and preserve lacZ but it seems a bit
complicated. Has anyone successfully used an anti-lacZ antibody to do this
in bone?

2) eGFP reporters (http://jaxmice.jax.org/strain/004077.html)
Jackson's website notes that this isn't suitable for immunohistochemistry
because expression is too low. Since bone is so autofluorescent, I really
doubt I'd ever see it.

I do have a chicken anti-GFP antibody, which seems to work really great in
paraffin sections with some mouse strains (col2.3-GFP) but doesn't seem to
work with others (CX3CR1-GFP) when I come in with a Dylight488 anti-chicken.
This seems to occur even though they seem to have similar brightness when
assayed by FACS... I'm not sure why. Has anyone used an anti-GFP antibody
for lineage tracing in bone or other fixed tissues?

If you're aware of any other strains or methods to accomplish this, I would
greatly appreciate your suggestions. There is a small chance that I may get
access to a Cryojane at some point in the future, and I would also welcome
comments on how feasible this would be using that system.

Thanks,
Adam