Re: advice on sectioning

From:"J. A. Kiernan" <>

On Tue, 5 Dec 2000, Vanessa Heim wrote:

> falling off the slides during the staining matter what
> protocol I use...we use precoated slides from surgipath, and I have even
> added addtional adhesive to a water bath to see if that would help the
> ... we use a cryostat to section

You'll get lots of advice on this!   I think your troubles are
due to putting unfixed cryostat sections into water. This will
instantly wreck the structure of everything. The changes are
invisible to the unaided eye, but with even a X10 objective it's
obvious that there's disruption of all the cells and distortion
in extracellular regions.

The usual procedure when cryostatting is to collect each section 
from the knife onto a slide. Some people prefer to collect onto 
a coverslip. The firm that sold you the cryostat should have 
provided some instruction on how to use this expensive
instrument. Knowledge, skill and lots of practice (which 
generates the skill) are needed to get large numbers of good 
cryostat sections.

Your adhesion failure should solve itself once you've learned 
the methodology of cryostatting. If you are using slides that
are sold for their special adhesive properties, do not also use
other adhesives (such as gelatin, albumen or a glue of uncertain
nature). These substances are likely to neutralize the adhesivity 
(if that's a word) of expensive bought slides, which are usually 
very good (though it's cheaper to make your own).

Water baths are used for floating sections of paraffin-embedded
specimens onto slides. Paraffin processing and sectioning also
require plenty of understanding, practice and skill, but not
as much as is needed to become competent with a cryostat. 

 John A. Kiernan,
 Department of Anatomy & Cell Biology,
 The University of Western Ontario,
 LONDON,  Canada  N6A 5C1

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