RE: [Histonet] vibratome sectioning of brain tissues

From:"Charles Scouten"

 
"Vibratome(TM)" is a registerd trademark of Vibratome company, formerly TPI.  You have a vibrating blade microtome from another company, not a Vibratome.

Thick Thin sections are most often caused by too shallow a blade angle.  Steeper usually helps.  However, thick thin can have other causes, such as the pedestal not rigidly locked to the instrument, or the blade not tightly locked down, or not a fresh sharp blade, or bad tissue quality.

If you have tried blade angles to no avail, try grasping the tissue pedestal, then the blade carriage, and see if anything is loose or wobbles.  

If nothing interesting found, I would worry about tissue quality problems due to cooking the tissue in warm gelatin. Or the wash in tap water.  Even partially fixed tissue can be damaged by tonicity challenges, although 25 hours in 4% formalin should mostly prevent that.  But rinse in a volume of PBS instead.

Many Vibratome users use no embedding.  Glue the fixed brain to the pedestal, cutting a flat surface to glue down, and begin sectioning.  The further from the pedestal you are cutting, the more flex in the system, and the more likely is thick thin, so try to block the brain down to the chunk you need by cutting off the ends far from the part you need.  

Let me know if any of this helps.


Cordially,
Charles W.  Scouten, Ph.D. 
myNeuroLab.com 
5918 Evergreen Blvd. 
St. Louis, MO 63134 
Ph: 314 522 0300 x 342
FAX  314 522 0377 
cwscouten@myneurolab.com 
http://www.myneurolab.com 
 

-----Original Message-----
From: histonet-bounces@lists.utsouthwestern.edu [mailto:histonet-bounces@lists.utsouthwestern.edu] On Behalf Of Bruijntjes, J.P.
Sent: Friday, March 10, 2006 10:20 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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