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 _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet