[Histonet] RE: Using Isopropyl alcohol in tissue processor (Courtney Cain)

[John Shelley]

 Hi Courtney,

I was wondering at what temperature you have your wax at because I think it could be that all the alcohol is not clearing out of the tissue and that is why you are getting some of that separation. Do you ever smell alcohol in the tissue that explode like you do when xylene has not completely cleared out. I am also working with the Excelsior and Isopropyl alcohol and found that my alcohol times needed to be a bit longer and the wax should probably be double to what you are doing now. You can email me and I can give you my protocols.

John Shelley
Histology Core Facility
Burnham Institute for Medical Research
8669 Commodity Circle
Orlando, FL 32819
email:jshelley <@t> burnham.org 

Message: 5
Date: Sun, 4 Jan 2009 20:55:01 -0600
From: "Courtney Cain" <Courtney.Cain <@t> pbrc.edu>
Subject: [Histonet] Using Isopropyl alcohol in tissue processor
To: <histonet <@t> lists.utsouthwestern.edu>
Message-ID: <B090B210273F854BBAB14241C4DD9DA4A8E702 <@t> pbrcas31.pbrc.edu>
Content-Type: text/plain;	charset="iso-8859-1"

 
I was wondering if anyone has tried Isopropyl Alcohol (2-propanol) as an alternative to xylene substitutes in their tissue processors.  We are attempting this method with the Thermo Excelsior to process mouse embryo, placenta, liver, and mouse and rat organs. Currently our animal tissue has been immersion fixed, but we have encountered extremely brittle and poor morphology of placenta and livers. A few livers and kidneys have pulled away from the wax as well. 
 
Here is our basic protocol that has been attempted with and without vacuum.
 
Formaldehyde                  2 hr        
Formaldehyde                  2 hr
Isopropyl Alcohol 70%    20min
Isopropyl Alcohol 90%    20min
Isopropyl Alcohol 95%    20min
Isopropyl Alcohol100%  20min
Isopropyl Alcohol100%  20min
Isopropyl Alcohol100%  20min
Wax                                       30min
Wax                                       30min
Wax                                       30min  
 
Thanks,
 
Courtney Cain
Research Associate
Cell Biology & Bioimaging Core
Pennington Biomedical Research Center
225-763-2653
caincm <@t> pbrc.edu
 

Message: 7
Date: Mon, 5 Jan 2009 07:13:41 -0800 (PST)
From: Rene J Buesa <rjbuesa <@t> yahoo.com>
Subject: Re: [Histonet] Using Isopropyl alcohol in tissue processor
To: histonet <@t> lists.utsouthwestern.edu, Courtney Cain
	<Courtney.Cain <@t> pbrc.edu>
Message-ID: <970626.67449.qm <@t> web65716.mail.ac4.yahoo.com>
Content-Type: text/plain; charset=iso-8859-1

Iso-propanol (2-propanol) is the most secure, direct and cheapest alternative to xylene. The only thing I will recommend you is to have the first paraffin bath made of paraffin and 2-propanol at equal amounts in order to reduce the solubility gradient between the last 2-propanol and the first paraffin steps from 9 to 4.5 Mega Pascals.
Other than that you will not only eliminate xylene but will reduce costs since 2-propanol is cheaper than ethanol and xylene.
Most of the Peloris tissue processors users?rely on?2-propanol as both dehydrating and "ante medium" agents.
To clean the tissue processor instead of xylene use a 2% aqueousus v/v solution of a laboratory strength dish washer soap, and you are set to have a tissue processing schedule free from xylene.Ren? J.

--- On Sun, 1/4/09, Courtney Cain <Courtney.Cain <@t> pbrc.edu> wrote:


From: Courtney Cain <Courtney.Cain <@t> pbrc.edu>
Subject: [Histonet] Using Isopropyl alcohol in tissue processor
To: histonet <@t> lists.utsouthwestern.edu
Date: Sunday, January 4, 2009, 9:55 PM

 
I was wondering if anyone has tried Isopropyl Alcohol (2-propanol) as an
alternative to xylene substitutes in their tissue processors.  We are attempting
this method with the Thermo Excelsior to process mouse embryo, placenta, liver,
and mouse and rat organs. Currently our animal tissue has been immersion fixed,
but we have encountered extremely brittle and poor morphology of placenta and
livers. A few livers and kidneys have pulled away from the wax as well. 
 
Here is our basic protocol that has been attempted with and without vacuum.
 
Formaldehyde                  2 hr        
Formaldehyde                  2 hr
Isopropyl Alcohol 70%    20min
Isopropyl Alcohol 90%    20min
Isopropyl Alcohol 95%    20min
Isopropyl Alcohol100%  20min
Isopropyl Alcohol100%  20min
Isopropyl Alcohol100%  20min
Wax                                       30min
Wax                                       30min
Wax                                       30min  
 
Thanks,
 
Courtney Cain
Research Associate
Cell Biology & Bioimaging Core
Pennington Biomedical Research Center
225-763-2653
caincm <@t> pbrc.edu
 
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End of Histonet Digest, Vol 62, Issue 5
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