Re: [Histonet] Re: cresyl violet staining vs. Haematoxylin staining {LONG}

From:John Kiernan

This is an answer to two Histonet questions asked
on 27th Jan 2006. It's addressed primarily to
Chengkang Zhang, who asked about staining neurons
with haemalum ("haematoxylin") or cresyl violet.
The other question, from MOHANA was about the
mechanism of Nissl staining by cresyl violet. The
answers for MOHANA are mostly in the next
paragraph but one. 

Assuming that you (Zhang) are using a haemalum
(alum-haematein): this stains the nuclei of all
cells, including neurons and neuroglia. There are
other haematoxylin-based stains that are very
selective for nuclei, ones that stain myelin, and
all sorts of others. Haemalum is the H in H&E.

Cresyl violet, on the other hand, is a cationic
("basic") dye that attaches to anionic
macromolecules. In the central nervous system,
nuclear DNA and ribosomal RNA (rRNA) are the only
such materials. DNA is in the nuclei of all cells:
neurons and neuroglia. All cell nuclei are stained
by cresyl violet, just as they are (though for a
different reason) by haemalum. RNA is present in
the cytoplasm of all cells, but neurons contain
much more rRNA than glial cells. In the cytoplasm
around a neuron's nucleus (the perikaryon), the
rRNA is often organized into stripes or granules,
characteristic of the type of neuron, called Nissl
bodies. (Messenger RNA and transfer RNA probably
doesn't contribute to the picture; that's another

Nissl didn't use cresyl violet, by the way; he
used methylene blue. (In the December 2005 issue
of Microscopy Today, on p.50, there's a historical
note about Franz Nissl.) 

Large and medium-sized neurons and the main glial
cell types can be identified from the appearances
of their nuclei (stained with either haemalum or a
cationic dye). Some small neurons have densely
staining nuclei and cannot be certainly
distinguished from oligodendrocytes in stained
sections. Among the granule cells of the
cerebellum or olfactory bulb, or in layer 4 of a
sensory area of the cerebral cortex, individual
cells cannot be categorized on the strength of
nuclear appearance. In such regions the
cytoplasmic Nissl bodies are not very helpful. 

A small interneuron has only a little stainable
cytoplasmic rRNA. Undisputed oligodendrocytes (in
white matter) also have subtle rings of rRNA
around their nuclei. This glial cytoplasmic rRNA
is distinct from the DNA subjacent to the nuclear
membrane, and it stains red (RNA) not blue-green
(DNA) with the ethyl green-pyronine method. I've
seen this myself, long ago, but cannot conjure up
the even older original refs. If your library has
"Methods in Brain Research" edited by P.B.Bradley
(Wiley, 1975), you may find the ref in Chapter 1. 

John Kiernan
Anatomy, UWO
London, Canada
Chengkang ZHANG wrote:
> I am currently using haematoxylin to stain nuclei of neurons, but
> someone told me that cresyl violet is better than haematoxylin. Would
> anyone tell me what the difference between those two staining and which
> one you prefer for nuclei staining?
> Thanks
> Chengkang
> --
> ===================================
> Chengkang Zhang Ph.D.
> Department of Pharmacology
> University of California, Irvine
> Irvine, CA  92697-4625
> Email:
> TEL:    (949)-824-1902 (lab)
> FAX:    (949)-824-4855
> ===================================

MOHANA wrote:
> i was wondering if any one can explain me that why does the cresyl violet stains only neurons and not glia????
>   mohana
> Mohana

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