[Histonet] HELP, Plastic sectioning

[Pamela Marcum]

At 01:57 PM 2/13/2006, Sunhwa Lee wrote:
>I am trying to do plastic sectioning with Fish brain and the section
>is shattered a lot.  I've been changing thickness 1-5um, new knife,
>and embedding materials (JB-4 and Technovit 7100), but not thing
>works. My slicing conditions are as follows.  Any suggestions, please.
>  In advance, thank you so much.
>
>1. Knife : High profile stainless steel knife
>2. Microtome: normal rotating Microtome (Leica 820-II)
>3. Thickness: 1-5um
>4. Cutting angle: 2-3degree
>5. Embedding material: GMA (Technovit 7100)
>6. Sample: fish brain
>
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Hi Sunwha,

You did not mention how you are fixing and dehydrating the tissues and that 
can affect sectioning also.  What is you schedule and how are you 
polymerizing?  Is it room temperature or 4C?  You thickness with the 
correct knife and tissue preparation is not the problem.

As you are using JB4 or Technovit 7100 it will still require a glass knife 
or tungsten carbide knife at the least for your best sections.  That may 
require a special knife holder for the older blade types as in 
pre-disposable.  I know Linda Jenkins has used disposable knives however, I 
believe they were the low profiles.  Plastics are simply too hard for most 
disposable knives and the knives will deteriorate too quickly for you get 
good sections routinely.  A standard microtome is usable just not 
recommended as they are not generally heavy enough to cut smoothly with 
very hard materials.  I have seen people with tons of experience section 
well on lighter microtomes and I have done some it is just much harder to 
get consistent sections.  Angle is determined by trying different positions 
and seeing what works best.  One angle is not good for all blocks.  I 
change angle sometimes when the block is larger or smaller and it is always 
while I face the block so I don't lose sections of my tissue.  Hope this helps.

Best Regards,

Pamela A Marcum
Manager, Histology Special Procedures
University of Pennsylvania
School of Veterinary Medicine
R.S. Reynolds Jr.  CORL
New Bolton Center
382 West Street Road
Kennett Square, PA 19348

Phone - 610-925-6278
Fax     - 610-925-8120
E-mail - pmarcum <@t> vet.upenn.edu