thick versus thin sections of PMMA

From:Gayle Callis <>

Ease of handling may be the biggest factor.  If you cut and grind a
section, you will lose a good portion wasted in grinding procedure. There
are also ways of doing tape methods to pick up sections (serial is
possible).  You should be able to grind a section thinner than 300 um, 100
um is not unheard of, even 70 um.  You need a good dial caliper to measure
thickness,  and automated grinder (this saves finger joints from permanent
damage!) after getting the thicker wafered section using an Isomet saw, 1
mm thick (1000 um) is possible, it takes a tidge of patience and skill to
get them, be sure you weight them after cutting to prevent curling before
mounting on slides. Some thin sections can be stained without removal of
PMMA, see next comments. 

Thin sections permit total PMMA removal, thicker 300 um sections would have
to be surface stained but that is spectacular when done well, especially
with acid etching on surface. After PMMA removal, staining methods are far
greater in number since you have rid yourself of hydrophobic plastic which
prevents penetration of large molecules in aqueous solution.    With
surface staining you are limited to low molecular weight stains (Stevenels
blue or Sandersons rapid bone stain, methylene blue/basic fuchsin,
toluidine blue, MacNeals tetrachrome, light green, van Gieson, or basic
fuchsin for counterstaining, etc.) 

Surface stained sections require a white plastic slide or white surface
under section for viewing with microscope where the light is diffused up
and around the section.  The thin microtomed sections permit the usual
transmitted light for normal viewing.     

At 03:29 PM 8/13/01 -0400, you wrote:
>Hello Histonetters,
>One of our post-docs has questions regarding sectioning of mouse 
>bodies embedded in PMMA.
>I appreciate your efforts in resolving these issues for our project.
>With any luck, this should help us out.
>1.  When using calcien staining to assess new bone
>growth of newborn mice, does thin sectioning (10
>micron) of PMMA have additional benefit over thicker
>polished ones (300 micron)? The spine is the area of
>main interest.
>2.  Is there any particular hazards or pitfalls
>relative to the above method of staining when making
>thin sections?
>3.  Are there any nice tricks to keeping 10 micron sections
>  of PMMA  from crimping or folding over on themselves, thus
>  making interpretation more difficult?
>4. I know that it is difficult to adhere thin sections of  PMMA
>to slides.  Does anyone know any special techniques for doing so
>Thin sections if taken will be made using a Polycut-E microtome
>  using a tc knife.
>Thanks,  Ryan
>Thanks for you help in answering these questions.
>John A. Baker
>The University of Michigan
>Orthopaedic Research Laboratories
>Histology Unit
>400 North Ingalls, G160
>Ann Arbor, MI 48109-0486
>Main lab office phone:734-763-9674
>Histology office:734-936-1635
Gayle Callis
Histopathology Supervisor
Veterinary Molecular Biology

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