[Histonet] manual tissue processing protocol

[Kim Donadio]

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 

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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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