[Histonet] manual tissue processing protocol

[Tyrone Genade]

Thanks


On Thu, Feb 27, 2014 at 5:50 PM, Kim Donadio <one_angel_secret <@t> yahoo.com>wrote:

> I agree with Renee here about testing one before. However those are thick
> and I'm supposing your fish have scales?  This also might slow the process
> down. My best guess is you're going to need a much longer processing time.
> And since you don't have vacuum I personally think that thickness is going
> to need a few hours. I've never processed a fish though take that for what
> it's worth. Good luck
>
> Sent from my iPhone
>
> On Feb 27, 2014, at 1:15 PM, Rena Fail <renafail2 <@t> gmail.com> wrote:
>
> > Hi,
> >
> > One mm thick is tiny, 2 mm small  1 cm is rather thick. Since you are
> > manually processing run one sample to test your protocol  or  2 if you
> want
> > to see  the difference between the shorter method and the longer.
> *protocol.
> > .Complete  dehydration is dependent on the volume, type of tissue and
> more
> > importantly, the thickness of the tissue. You can check the tissue before
> > infiltrating with paraffin.to <http://paraffin.to> see if it has
> > cleared, If it appears transparent (like a stained glass window.) proceed
> > with paraffin infiltration. *
> > *Rena Fail*
> >
> >
> > On Wed, Feb 26, 2014 at 2:02 PM, Tyrone Genade <tgenade <@t> gmail.com>
> wrote:
> >
> >> Hello,
> >>
> >> I need some input on a manual tissue processing protocol I have
> received.
> >>
> >> (5 columns: reagents; tiny to large tissues...)
> >>
> >> Reagents\embryoes\small tissue\medium tissue\large tissue
> >> 70% EtOH\30 min--1hr\1 hr--ON\2hr--ON\4hr--ON
> >> 95% EtOH\30 min--1hr\1 hr\1hr\2hr
> >> 95% EtOH\30 min\30 min\1 hr\1 hr
> >> 100% EtOH\30 min\1 hr\30 min\1 hr
> >> 100% EtOH\10 min\30 min\30 min\1 hr
> >> toluene\30 min\1 hr\1hr\1 hr
> >> toluene\none\30 min\30 min\1 hr till clear
> >> parafin infiltration\1hr all
> >> parafin infiltration\1 hr\1 hr\2hr\2hr
> >> EM400\1 hr\1hr\1hr\2+ hr
> >>
> >> I would be replacing the toluene with methylbenzoate. Are there any
> >> anticipated issues? Is this a good protocol so far as producing good
> >> quality tissue for sectioning and analysis?
> >>
> >> I am not sure what is meant by "small" and "large" tissue. My fish are
> >> about 5 cm in length and about 1 cm thick. Would they be classed as
> small?
> >>
> >> Is it necessary using two different types of paraffin : Surgipath
> >> infiltrating paraffin Cat # 01400 (4-5 lb/bag) and Surgipath embedding
> >> paraffin? For the manual infiltration: would a beaker of paraffin on a
> >> 60oC hotplate and stirrer (on low) be OK?
> >>
> >> I would be fixing with PFA and decalcidying with 30% EDTA. Might
> refixing
> >> in the PFA after decal be a good idea to make sure the tissue integrity
> is
> >> maintained? I would probably stored fixed, decalsified samples in 70%
> EtOH
> >> until I can process fully. Any one think there would be complications
> from
> >> doing this?
> >>
> >> Thanks
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> >>
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>



-- 
Tyrone Genade
http://tgenade.freeshell.org
email: tgenade <@t> gmail.com
tel: +27-84-632-1925 (c)
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