RE: [Histonet] whole brain in paraffin

From:Sharon Allen



Hi,
I have done whole mounts but not for about 6 years (the neuropathologist
that used to like them has left - hurray!!). I am including our method for
processing the block. I hope this is of some help, please email me if you
have any questions. 
Sharon Allen
HSC Winnipeg, MB, CA
sallen@hsc.mb.caHAND 

*Sorry about the uppercase, just the way our manual was typed.

PROCESSING WHOLE BRAIN SPECIMENS

HAVE SPECIMEN CUT AT A THICKNESS OF 1.5 TO 2.5 CM.   ANY THINNER AND THE
SPECIMEN MAY CURL, MAKING IT DIFFICULT TO EMBED FLAT.  

THE PROCESSING TIMES DEPENDS MAINLY ON THE SIZE AND THICKNESS OF THE TISSUE.
THE TIMES USED IN THE FOLLOWING LIST WAS FOR A SPECIMEN WHICH WAS 2 - 3 CM
IN THICKNESS AND 10 CM IN WIDTH.  THE PROCESSING WAS SUCCESSFUL.

THE TISSUE CAN BE LEFT IN FORMALIN, WAX,  70% AND 95% ALCOHOL , FOR AN
INDEFINITE TIME WITHOUT HARM.

*CHANGE THE SOLUTIONS A FEW  TIMES PER DAY.
	
	FORMALIN-----------------------------------APROX. 1 WEEK
	70% ALCOHOL------------------------------OVERNIGHT	
	70% ALCOHOL------------------------------OVERNIGHT

	95% ALCOHOL------------------------------OVERNIGHT
	95% ALCOHOL------------------------------OVERNIGHT
	95% ALCOHOL------------------------------OVERNIGHT

	100% ALCOHOL----------------------------OVERNIGHT
	100% ALCOHOL----------------------------OVERNIGHT
	100% ALCOHOL----------------------------OVERNIGHT

	CHANGE TO XYLOL, CHANGING SOLUTION EVERY HOUR AT FIRST.
	KEEP CHECKING TO SEE IF IT IS CLEARING.  (Hold section up to a
light)
	THE TISSUE WILL BECOME MORE TRANSLUCENT AND A LIGHTER COLOR AS IT
CLEARS. THE CLEARING WILL TAKE PLACE FROM THE OUTSIDE, INWARD; MAKE 	SURE
THE CENTER IS CLEARED.
	XYLOL WILL HARDEN THE TISSUE, SO CARE MUST BE TAKEN WITH THE
ENDPOINT AND TIMING.
	
	WHEN THE CLEARING IS COMPLETE, PUT THE SPECIMEN IN WAX, CHANGING THE
SOLUTION  OFTEN.
	THE VACUMM OVEN CAN BE USED AT THIS POINT.  SET IT AT THE MELTING 
	POINT OF THE WAX AND AT 15 LBS. PRESSURE FOR 1 HOUR AT A TIME, THEN
CHANGE THE WAX, REPEATING THE INFILTRATION UNDER VACUMM. 
	THIS PROCESS CAN TAKE 2 TO 5 DAYS, (INFILTRATION IN THE WAX CAN NOT
HARM THE TISSUE)  FREQUENTLY CHANGING THE WAX, AND LEAVING THE 	SPECIMEN IN
A 54C OVEN OVERNIGHT OR FOR THE WEEKEND, IF NECESSARY.

      THE SPECIMEN IS THEN EMBEDDED AND ATTACHED TO THE CHUCK USED FOR THE

      SLEDGE MICROTOME. HEAT THE CHUCK, PLACE IT ON THE BACK OF THE BLOCK
AND  
      THEN SUBMERGING IT INTO A SINK FILLED WITH ICE COLD WATER.

      CUT SECTION ON THE SLEDGE MICROTOME AT 6 TO8 MICRONS.

-----Original Message-----
From: histonet-bounces@lists.utsouthwestern.edu
[mailto:histonet-bounces@lists.utsouthwestern.edu]On Behalf Of Nancy
Lemke
Sent: February 16, 2006 7:25 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] whole brain in paraffin


Good Morning Histonet,
I would like some information about the preparation and sectioning of 1-2cm
slabs of human brain.  The cutting will be done on an old Reichert Tetrander
(sliding) microtome with a giant steel blade.  If anyone who has experience
with this sort of sectioning would email me I would love to ask some
questions.
Thank you in advance,
Nancy Lemke
Research Coordinator
Hermelin Brain Tumor Center
Henry Ford Hospital
Detroit

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