I am having some problems with a subset of antibodies on adult mouse DRG. These antibodies work great on embryonic tissue, but on adult tissue they are failing.
The only methodological changes between embryonic tissue and adult is a shorter (1-2 hours tried) fixation time (4% PFA in PBS) for isolated adult DRG compared with 3-4 hours for an E16.5 embryo.
I am using indirect immunofluorescence staining.
I am having issues with antibodies against proteins located in the nucleus, as well as those in the cytosol.
Any help would be appreciated before I start an intensive empirical investigation on the best immunostaining protocol for this tissue.
I'm trying to get an immunostaining protocol to work in adult DRG myself at the moment (rat though). My protocol has me fix them with 4 % PFA for only 15 min. Why is the fixation so long for embryonic DRG?
Sorry for the confusion. We are fixing the whole embryo for that long, we don't dissect out the DRG in the embryos. Mainly because we don't have to, they are small enough that we can get enough transverse sections of the whole embryo on a slide. Not so easy in the adult, so the DRG are dissected out.
Do you use Triton X-100 to permeabilize the membrane? Formaldehyde treatment does cause membrane perforations, but probably not enough in the shorter fixation time used on the adult DRGs. You can find our protocol used for DRG immunostaining in the link below.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3393828/
Catherine Elizabeth Moss After you treat your DRG tissue in 4% PFA for 1-2hrs, do you leave the DRG in 100% ethanol? Also do you have to intracardially infuse PFA before dissecting the DRG out of the animal?
I am very new to this method and its hard to find these initial steps before the cryosectioning and staining.
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