[Histonet] Acetone fixing and tissue damage

[Gayle Callis]

Dear Bernice, 

This has always been my favorite solvent fixative for murine CD markers and
some other markers/antigens but it can't be used for human CD4 and CD8 IHC.
These human  markers do not tolerate the alcohol component  and won't stain.
The late Dr. Chris van der Loos imparted this bit of information after I
recommended he should try it.   I think most people do human CD4 and CD8 on
FFPE tissues now but other human markers might be similar to the CD4 and CD8
and not stain after acetone/absolute ethanol.     

Acetone/alcohol in this ratio has been by fixative of choice for the mouse
and rat CD markers but I used it at RT, not cold.  The alcohol should be
absolute ethanol.     With this in mind,  a recommendation is do a  fixation
panel with different fixatives to optimize for any given antigen.     

Air drying the fresh tissue frozen section is a must though.     

Gayle Callis 
HTL/HT/MT(ASCP)  

  

-----Original Message-----
From: Bernice Frederick [mailto:b-frederick <@t> northwestern.edu] 
Sent: Thursday, March 12, 2015 11:49 AM
To: gayle.callis <@t> bresnan.net; histonet <@t> lists.utsouthwestern.edu
Subject: RE: [Histonet] Acetone fixing and tissue damage

We use a 3:1 ration of cold acetone/100% ETOH to fix frozens for IHC. Slides
are always air dried first to remove any moisture.
Usually 75 mls acetone,25 mls alcohol.

Bernice Frederick HTL (ASCP)
Senior Research Tech
Pathology Core Facility
Robert. H. Lurie Cancer Center
Northwestern University
710 N Fairbanks Court
Olson 8-421
Chicago,IL 60611
312-503-3723
b-frederick <@t> northwestern.edu


-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Gayle Callis
Sent: Thursday, March 12, 2015 12:02 PM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Acetone fixing and tissue damage

You wrote:   

 

Hi Everyone.

 

When I fix my cryosections  in acetone,  I am using HPLC grade 99.9% for 10
minutes at -20C.

 

Would the Histology grade 99.5% be less damaging to them?

 

Higher H20 content, i.e. less than 99.5% apparently is also very bad.

 

With the HPLC grade I often get tissue damage, the tissue also floats off
the slide causing a stringy effect.

 

Fixing with 4% p-formaldehyde or 100% Methanol, prevented the antibody from
recognizing the Nuclear Antigens.

 

Looking for advice,

 

Patrick.

Patrick Lewis

Research Associate II Bench

Seattle Childrens Research Institute

206-884-1115

 

****************************************************************************
*********************

HPLC grade acetone is not necessary plus very expensive.  Use ACS Certified
Reagent 99.5% grade, not histology grade, which you can buy in gallon size.
Maybe what you are calling histology grade is the ACS Certified Reagent
grade but  "Histology" grade implies a practical grade of acetone which is
not a pure as the ACS certified Reagent grade.   Also, 4°C acetone works
just as well.    If you are storing your acetone (in a staining jar) inside
the cryostat to maintain a 20°C temperature, don't!!!  If your staining
container tips over, you will ruin your cryostat!!   Hopefully you are using
high quality plus charge slides?        

 

We had frozen sections come off a plus charge slide after single 4°C acetone
fixation on occasion.  You can prevent frozen section loss is a Double
Acetone fixation that also increases permeabilization.     An IHC guru gave
me this hint years ago and was given to her by a company selling
immunostaining products.   A small fan will be your best friend for RT air
drying and/or evaporating acetone.  However we air dried all  FS were dried
at RT for 30 minutes minimum  or longer before fixation.  By air drying, you
get rid of the water.  You can put your FS in front of a small fan, or
inside a hood for faster drying, and never store just cut FS in the cryostat
where water condensation occurs when you take them out of cryostat
environment to RT.    DRY  frozen sections were the rule in our lab before
any solvent fixation.      

 

1.   Air dry frozen section at RT for 30 min 

2.   Fix FS in  4°C acetone for 10 minutes

3.   Air dry FS for 15 minutes to evaporate acetone

4.  Return FS to 4°C acetone for 10 minutes

5.  Air dry to evaporate acetone 

6.  Proceed to immunostaining 

 

Gayle Callis

HTL/HT/MT(ASCP)

 

 

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