Re: [Histonet] 20 micron sections Cresyl Violet Acetate stained

From:"John A. Kiernan"

There were 3 reasons for using 20um sections
(though 6um sections were also used in the paper
that you quote).

1. The spinal cords of interest were from people
who had died of ALS, and motor neurons were not
numerous. A thick section has more cells in it
than a thin one.(The control subjects had plenty
of motor neurons, of course, but the sections had
to be the same thickness for quantitative
comparisons.)
2. 20um was the thickest that was easily handled
with paraffin sections. For neuroanatomical work,
frozen sections are commonly cut at 40-80um for
Nissl staining. 
3. You need to include as much neuron as possible
in the section, to show the shape and size.

We did not have trouble with sections coming off
the slides, which were coated (subbed) with
chrome-gelatin.

There was no difficulty with either the myelin
stain (iron-eriochrome cyanine R; Page's method)
or with the Nissl counterstain. Neutral red was
used because it contrasted well with the blue
myelin. If you're not interested in myelin you can
use any cationic dye as a Nissl stain. Cresyl
violet can be quite a fiddle; make sure you have a
certified batch of the dye. Neutral red or
toluidine blue (0.5%, pH 3.5 to 4) is easier to
use. H&E is not suitable for staining neurons. 
-- 
-------------------------------
John A. Kiernan
Department of Anatomy and Cell Biology
The University of Western Ontario
London,   Canada   N6A 5C1
   kiernan[AT]uwo.ca
   http://publish.uwo.ca/~jkiernan/
   http://instruct.uwo.ca/anatomy/530/index.htm
_______________________________
Patrick Laurie wrote:
> 
> Hi Histonet,
> 
> My group is interested in doing an anterior horn alpha motor neuron
> count of ALS pts vs control.  There is considerable literature about
> these techniques, which has been most helpful, but the devil is in the
> details.
> 
> Most papers use 20um sections of the ventral horn stained with cresyl
> violet acetate, or iron-chromoxane cyanine R method for myelin
> (counterstained with .5% neutral red) (Dr. Kiernan's 1991 paper), or one
> of many other methods for staining.  I have 3 questions.  First of all,
> a technical question, how are 20um sections kept on the slides during
> staining?  I have done some experimenting, and found it very difficult
> to keep them on.
> 
> Secondly, how do the thick sections stain?  The cresyl violet using will
> not differentiate very well (using the regressive version), and the H&E
> is too dark, I have yet to use any other method.
> 
> And lastly, why 20um?  Conventional neural histology seems to be done
> thicker, but usually not that thick.  What would be the benefit for
> having sections that thick?
> 
> Thanks Histonet,
> 
> Patrick Laurie, HT (ASCP)
> Neurogenomics Laboratory
> Benaroya Research Institute
> 1201 9th Ave
> Seattle, Wa  98101
> 
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