RE: [Histonet] breast processing

From:Kemlo Rogerson

Prolonged fixation, thinner blocks, and extended processing. Unfixed fat
doesn't process very well mainly in part I guess as you can't cut the blocks
thin enough; there's some strange equation concerning time for fluids to
penetrate, but basically the thicker the block then the time of penetration
rises exponentially.

You could also try going back into a dehydration fluid after 'clearing' in
xylene as that is a fat solvent; so you remove the fat and expose more
tissue to be dehydrated.  

Kemlo Rogerson
Pathology Manager
Ext  3311
DD   01934 647057
Mob 07749 754194

"Following the line of least resistance makes both men and rivers crooked"


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-----Original Message-----
From: Featherstone, Annette [mailto:AFeatherstone@KaleidaHealth.Org] 
Sent: Tuesday, April 04, 2006 2:26 PM
Subject: [Histonet] breast processing

Does anyone have any suggestions for better tissue processing for fatty
tissue such as breast? We are experiencing insufficent dehydration, clearing
and infiltration. 

Annette Featherstone HT/MLT

-----Original Message-----
[]On Behalf Of
Sent: Sunday, April 02, 2006 13:01
Subject: Histonet Digest, Vol 29, Issue 2

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Today's Topics:

   1. My frustrating experience with double fluorescence	staining!
      (Chengming Wang)


Message: 1
Date: Sat, 01 Apr 2006 23:25:00 -0600
From: "Chengming Wang" 
Subject: [Histonet] My frustrating experience with double fluorescence
Message-ID: <>
Content-Type: text/plain; charset=US-ASCII

Dear All,

I am new with histology, and  posted yesterday a message of: Help
needed!!!  Immunofluorescence double staining with mouse lung.  Thanks a
lot for the several helpful responses.  As asked, I am here putting on
my concise protocols for your check:

Sampling:  Cut a small cut along the mouse trachea, injected neg 50
(similar to OCT). Then quick freeze with liquid nitrogen, and the stored
@ -80; After section with 6 um, slides were fixed with  icy acetone for
5 minutes. Quick dry, and then stored @ -20 till use.

Staining: 1. Washing 3 X 5 mins; Blocking Buffer (10% Rabbit serum; 5%
BSA; 0.1% Tween 20) for 1 hour at room temperature;
                2. Tapping away the blocking buffer, and applied first
antibody for 1 hour; then wash 3 x 5 min'
                3. 2nd antibody 1 hour; washing 3 x 5min;
                4. Mounting media, read slides. Count stained with
DAPI-mounting media.
                 5. For double staining, I did the same thing, except
using mixed first antibodies and mixed second conjugated antibodies.

Designing: Stain 1: Rabbit anti-mouse antibody, and donkey anti-rabbit
conjugated with Alexa Fluor 488;
                Stain 2: Rat anti-mouse antibody, and donkey anti-rat
conjugated Alexa fluor 594;   

My problems:  The individual staining signals are good. But there is
some match color between two channels. For example, I could see weak and
similar green signal when I applied Alexa 594. Basically, I could see
the weak but true alveoli structure of mouse                            
                       Then I performed double staining, I could see
lots of signal match between red and green channels, which should not be
true. In this way, I could see basic alveoli structure of mouse         
                        lung in both green and red channels. By the way,
I tried to red the slides before staining and after blocking, the
autofluorescence is very very weak.

I am really frustrating with this situations, and please feel free to
tell me how I could figure out this problem. Any of your suggestions and
comments are very much appreciated.


Chengming Wang 
DVM, M.S., Ph.D. 
Department of Pathobiology
College of Veterinary Medicine
Auburn University
264 Greene Hall
Auburn, AL 36849-5519

Voice: (334) 844-2624


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