RE: Immuno Staining and Xylene Substitute

From:Gayle Callis <>

IF "some people have had to deparaffinize in three changes for 20 minutes
each" I think more specifics are needed here.  Terpenes?  Single aliphatic
hydrocarbons (Propar or Clearite 3, others?  Specific brands would be helpful.

By reeking havoc, does this mean weak staining, spotty staining, etc., etc.
What are other problems, other than background staining.  There are more
variables in this equation than just xylene substitutes. Fixation,
processing in general,  paraffins used, temperatures in processing??
Retrieval?? etc

There is no doubt complete removal of paraffin is essential, and increasing
time in changes is a factor, but having cleaner reagents (rotating out
excessively used solvent) could avoid some of the long time changes to
avoid paraffin carryover.  

Just curious here 


>Date: Wed, 18 Oct 2000 11:15:35 -0500
>From: "Nocito, Joseph" <>
>Subject: RE: Immuno Staining and Xylene Substitute
>To: "''" <>, 
>in my experience with reference material, specimens processed with xylene
>substitutes justed reeked havoc with immunos.  I have experienced a general,
>non-specific staining from one lab. When I asked them what they were using,
>they said they were using a xylene-substitute (I can''t remember the brand)
>Why this happens, I don't know the actual chemical reaction.  I do know that
>if you are deparaffinizing with a xylene substitute, you need to increase
>the number of changes in addition to increasing the times.  I've heard that
>some people have had to deparaffinize in three changes for 20 minutes each.
>Hope this helps
>Joe Nocito, B.S., HT(ASCP)QIHC
>Histology Supervisor
>Christus Santa Rosa Hospitals
>San Antonio, Texas 
>> -----Original Message-----
>> From: []
>> Sent:	Wednesday, October 18, 2000 10:26 AM
>> To:
>> Subject:	Immuno Staining and Xylene Substitute
>> Hi everyone !
>> Has anyone heard or read anything with regard to poor antigenicity when
>> tissues are cleared using Xylene substitutes during processing or
>> staining?  Someone told me that they were told not to use xylene
>> substitutes of any kind for tissue processing or for the deparaffinization
>> and clearing steps in the staining process because it would effect immuno
>> staining negatively. They did not have details as to how, what or why.
>> Please enlighten me if you know anything with regard to this.
>> Thank You,
>> Mark Lewis 
Gayle Callis
Veterinary Molecular Biology
Montana State University
Bozeman MT 59717-3610
406 994-4705
406 994-4303

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