Re: Question (Hoechst)

From:"J. A. Kiernan"

Lynn Gruman wrote:
> Does anyone have a protocol for the Hoecht stain? I would 
> appreciate any information you can provide.

First off: Don't put "Question" as an email Subject.
For this question, "Hoechst" or even "Hoecht" would 
be OK. 

I think you probably mean Hoechst - the name of a 
drug company that also happens to make some cationic 
benzimidazole fluorochromes that bind selectively to
DNA with an accompanying increase in fluorescence.
They have such names as Hoechst 33258 (= bisbenzimide),
Hoechst 33342, and Hoechst S769121 (= nuclear yellow). 
These fluorochromes are commonly applied to cultured

They are very easy to use. For example, a solution
of bisbenzimide, 0.5 microgram per ml in a buffered
saline at pH 7.0-7.4, kept in a cool, dark place,
is stable for months (probably years). 

To use it:
  Fix the cultured cells in anything that 
preserves DNA (an acetic-alcohol does the job 
  Rinse in the same buffered saline or balanced 
salt solution used to dissolve the fluorochrome
  Stain in the fluorochrome solution for 15-60 
mins (not critical, but worth trying a few 
different times).
  Wash in 3 lots of the buffered saline.
  Coverslip, using a 50:50 mixture of glycerol
and the buffered saline.

Absorption is in the near UV (350-365 nm) and
emission is blue (460-480 nm). Any filter or
dichroic mirror block that allows broad band
UV excitation will do. The spectral properties
are similar to those of dansyl, coumarin or
cascade blue labels. That means you can't use
a fluorescence microscope that runs off a
quartz-halogen bulb (OK for fluorescein). 

If a halogen lamp fluorescence microscope is
all you have available, you'll need to use a 
different fluorochrome such as acridine
orange (CI 46005; 1 mg/ml at pH 6), or quinacrine
hydrochloride (0.25 microgram/ml at pH 7.2).
YOYO-1 iodide (a Molecular Probes product,
but not a trade secret), a cyanine dye, is 
sold as a solution in DMSO that must be diluted 
and used following the supplier's instructions. 
It's a modern alternative to the older
cationic fluorochromes; it is excited by blue
and emits green. The dyes mentioned in this
paragraph will stain both the nucleic acids.

If selectivity for DNA is important for you,
and you don't have an instrument that can look
at the Hoechst fluorochromes, consider ordinary
light microscopy and histochemistry. The
Feulgen method stains DNA a magenta colour.
The Feulgen reagents are much cheaper than Hoechst
dyes, YOYO-1 and other fashionable fluorochromes.

With Feulgen you don't need a fluorescence 
microscope to see that the DNA is in cell nuclei.
But if you must use one the stained nuclei are 
fluorescent, with a pleasing orange-brown
emission. The Feulgen reaction is 100% specific
for DNA. Most of the cationic fluorochromes,
especially the Hoechst dyes, are critically
dependent on pH for DNA specificity. 

The bottom line is: Go to the library. All the
stuff in the above paragraphs is in all the
histochemistry textbooks. Whatever you do,
don't stain your cells using this email as
your authority. 

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

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