[Histonet] manual tissue processing protocol

[Rena Fail]

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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