RE: [Histonet] vibratome sectioning of brain tissues

From:"Monfils, Paul"

I often section rat brains on the vibratome, and I must say my preparation
is far simpler than yours.  After fixation in formalin I rinse briefly in
PBS, transfer the brain to 3% agarose at 37 degrees for 15 minutes, then
place in 6% agarose in a plastic embedding mold and transfer to the
refrigerator (4 degrees) until fully gelled (about 10 minutes).  I remove
the block from the mold, trim its base by hand with a disposable microtome
blade to ensure a flat surface, blot that surface dry, and cement it to a
metal block with cyanoacrylate glue using gentle pressure for about a
minute, then place it in the block holder and begin sectioning.  The whole
process from fixed specimen to start of sectioning takes about 40 minutes.
Vibratome specimens don't have to be infiltrated.  They only have to be
physically supported by the surrounding medium. I use the 3% agarose
treatment in order to create a better bond between the embedding medium and
the surface of the tissue.

I don't like gelatin.  It doesn't yield as rigid a block as agarose, even at
higher concentrations.  I use a 20 degree knife angle; a fairly high
amplitude (7-8 out of a possible 10); and a slow speed (2-3 out of a
possible 10).  The water bath contains PBS with ice.

Thick/thin sectioning has three likely causes.  First, the block is
insufficiently rigid, that is, of too low a density;  second, the agarose
block is not firmly bonded to the block holder surface; or third, the blade
extends too high above the blade holder, so that the blade flexes as it cuts
through the block.  This causes the blade to dig into the block, forming a
thicker section, which results in the next section being too thin, because
some of the tissue that belonged to the second section went into the first
section.

Regards,
Paul Monfils

> ----------
> From: 	histonet-bounces@lists.utsouthwestern.edu on behalf of
> Bruijntjes, J.P.
> Sent: 	Friday, March 10, 2006 8:19 AM
> To: 	histonet@lists.utsouthwestern.edu
> Subject: 	[Histonet] vibratome sectioning of brain tissues
> 
> Hi all
> 
>  
> 
> A colleague of me works with rat brains and tries to prepare vibratome
> section but with hardly no result.    
> 
> Main problem is thin/thick sections. 
> 
>  
> 
> This is what she has done with the tissues ect: 
> 
>  
> 
> Brain tissue: perfused with and fixed pre-embedding in 4% Formaline
> 
>  
> 
> Embedding procedure: 
> 
> -during 24 H hemispheres were washed in running tap water
> 
> -during the following 24 H hemispheres were infiltrated with gelatin (from
> bovine skin 
> 
>   type B) solution, 12.5% w/w at 37║
> 
> -during the next 24 H hemispsheres were infiltrated with gelatin, 25% w/w
> at 37║
> 
> -after coagulation (approx 30 min) at 2-10 ║ gelatin blocks were further
> fixed during 4 
> 
>  days in 4% formaline (2-10 ║). 
> 
>  
> 
> A (new) vibratome, HM 650 V, was used for sectioning. 
> 
> Intended section thickness: 35 Ám
> 
> The buffertray was filled with PBS and crushed ice
> 
>  
> 
> She have tried many different combinations of the following settings:
> 
> -knife angle: 10-40 ║ (approx)
> 
> -frequency: 30-100 Hz
> 
> -amplitude: 0.3-1.2 mm
> 
> -speed: 1.5-5.0 mm/s
> 
>  
> 
> She is running out of ideas. Suggestions are very welcome!!! 
> 
>  
> 
>  
> 
> Joost Bruijntjes
> 
> TNO Quality of Life
> 
> Toxicology and Applied Pharmacology
> 
> Zeist
> 
> Holland
> 
>  
> 
>  
> 
> 
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