[Histonet] Re: Mast cells in spinal cord

[John Kiernan]

Dear Dr Cun,

You need to do some more homework! You probably found my name
because of two publications:
Campbell, D. J. and Kiernan, J. A. 1966. Mast cells in the
central nervous system. Nature 210, 756-757. 
Kiernan, J. A. 1976. A comparative survey of the mast cells of
the mammalian brain. Journal of Anatomy 121, 303-311.

These relate to mast cells in the normal CNS (which occur in only
a few species, if you exclude the ones in connective tissue
around blood vessels). In central nervous tissue, 
in contact with neurons and glial cells, mast cells occur only in
the habenular nuclei and nearby dorsomedial thalamus, in some
Insectivora and "lower" primates (tree-shrew, loris). 
In the 1990s Zhuang, Silver and others published a series of
papers about mast cells in the medial habenular nucleus of birds
- the place where they are most abundant in insectivores and
prosimians. 

To the best of my knowledge, mast cells do not occur in the
normal spinal cord of any vertebrate animal. Is your research
with the normal or diseased spinal cord? 

Mast cells are abundant in the dura mater. All research with mast
cells begins with a reading of Hans Selye's book "The Mast Cells"
(1963). Have you consulted this book? It contains the answers to
most of your questions. The fixation and staining problems had
all been nailed down by 1963. For most species you are wasting
your time trying to find mast cells in frozen/cryostat sections.
The tryptase and chymase activities do not exist in all species.
Rodents differ from humans and dogs. 

Discharged mast cell granules
 may hang around and be stainable as extracellular dots (rat), or
they may quickly dissolve and contribute to anaphlaxis (dog,
guinea-pig, some people). All this was known 40 years ago. 

John Kiernan
Anatomy & Cell Biology
London, Canada.
______________________________________
clcun <@t> itsa.ucsf.edu wrote:
> 
> Dear Dr. Kiernan,
> 
> I am trying to study mast cells in the CNS of mice and your name has popped
> up on several occasions.  I have a few questions regarding proper prepping/
> staining of mast cells and would greatly appreciate your expertise on this
> matter.
> 
> I would like to locate mast cells in the spinal cord using several stains:
> toludine blue, chloroacetate esterase, cKIT (ACK45) immunohistochemistry,
> tryptase and chymase enzyme histochemistry.
> 
> 1. What fixative is suitable for these stains?  We normally perfuse our
> animals with 4% paraformaldehyde and cryoprotect our tissues in 30%
> sucrose, but we’ve heard that this may not work for some of these staining
> protocols (especially, cKIT immuno and tryptase/chymase enzyme histo,
> which some have recommended using frozen tissues).  We would like to
> minimize usage of different fixation methods, so it would be great if we could
> get away with using paraformaldehyde, but only if it will give us good
> preservation of mast cells.
> 
> 2. What is the best way to embed our tissues?  After fixation, we usually
> cryoprotect with sucrose and block our tissues in OCT, but some people
> have suggested paraffin embedded tissues.  Unfortunately, our lab is not
> equipped to do paraffin embedding.  Would we be able to get good sections
> with our cryoprotected OCT blocked tissues?
> 
> 3.  Which orientation would be best to see the distribution of mast cells
> in the
> spinal cord, longitudinal or cross-section?
> 
> 4. Of the above stains that I’ve mentioned, which one do you think is best, in
> terms of specificity for mast cells, ease, etc.
> 
> Any comments and suggestions you can give would be greatly appreciated.
> Thank you in advance for your time.
> 
> Sincerely,
> 
> Christine Cun
> Department of Neurosurgery
> University of California, San Francisco