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> I am currently doing a post-doc in toxicology. My mentor is currently putting
> together a proposal that will include flow cytometry. We are both new to the
> field. We would like to attempt to identify pre-neoplastic hepatic cells at
> various points following exposure to toxicants. The toxicants are known to
> cause liver tumors in mice after chronic exposure. We would like to use markers
> that we have used in the earlier studies with immunostaining: c-myc, c-jun,
> TGFa and TGFb. We will harvest at least 30 liver samples on any given day. Is
> there a preferred method for sample storage? And for dissagregation of the
> tissue into cells? I have read the papers dealing with paraffin embedded
> samples, is this digestion procedure suitable only for collection of nuclei for
> DNA work?
>
> I would appreciate any advice that anyone has time to offer.
>
> Thanks in advance,
>
> Linda J. Graeter, Ph.D.
>
Difficult to give any specific advice here, but what we have been
doing is to Fine needle aspirate tumours and store the aspirate in
DMSO/serum freezing mixture. It is then stored either at -80C or in
liquid nitrogen(when the -80 freezer fails, curse it!) until required. We
have stained for a wide range of oncogene markers by washing out the
DMSO, fixing as appropriate and staining. For DNA there are two possible
courses: either use part of the aspirate by Vindelov's trypsin/detergent
method(good resolution) or add propidium iodide to your sample after
staining and display your cell marker in relation to cell cycle.
FNA samples seem to need little in the way of disaggregation: we
normally do not need to do any more than vortex the tube before running.
Hope this helps.
Eric Miller, ICRF Medical Oncology Unit, Edinburgh UK
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