Re: solochrome cyanine for GMA (rather long)

From:"J. A. Kiernan"

There is a paper:
Pierce M, Rodriquez M (1989) Eriochrome stain for 
myelin on osmicated tissue embedded in glycol
methacrylate. J. Histotechnol. 12:35-36. 

The procedure is as follows. I have not
tried it myself. More details are given
in the publication cited above.

1. Slides --> 1% periodic acid, 10 min.
2. Wash, running tap water 5 min.
3. Stain (see below) 40-60 min.
4. Running tap water 5 min.
5. Slides --> 10% iron alum until background
   is destained (1 to 2 min).
6. Running tap water 5 min.
7. Slides --> borax-ferricyanide (2 min).
     (Borax 1G, K3Fe(CN)6 1.25G, water 100 ml)
8. Running tap water 5 min.
9. Counterstain: 1% cresyl violet (6 min)
10. Distilled water 2 changes, then stand
    in 3rd change for 2 min.
11. Differentiate the cresyl violet in 
    89:10:0.5 alcohol:chloroform:acetic acid,
    until Nissl bodies are distinct .
12. Distilled water 2 changes, then stand
    in 3rd change for 2 min.
13. 95% alcohol, 2 changes, quickly.
14. Air dry.
15. Xylene, 2 changes, then coverslip
    with permount

Staining solution.
Stock: Eriochrome cyanine R (CI 43820) 1.5G
       Sulphuric acid (conc.) 2..5 ml
         Add acid to dye powder in a conical
       flask. Swirl and stir with glass rod
       until fizzing stops, then add
       Water   100 ml.
Working: Stock solution 16 ml
         Water  64 ml
         4% iron alum solution  20 ml.

The dye is CI 43820, Mordant blue 3. It has other
names including chromoxane cyanine R and solochrome
cyanine R. In chemical catalogues it is most often
called eriochrome cyanine R because analytical
chemists call it that. The 10th edition of Conn's
calls the dye eriochrome cyanine R too. 
Notes (from JK). I have used this dye in various
methods and done some research on its mechanisms of
action. See J. Microsc. 134: 13-23 and 25-39 (1984).
I can attest that the method of dissolving described
by Pierce & Rodriguez (and also by Page, 1965 and
in other papers) is not necessary. Just dissolve it
in diluted sulphuric acid. 

I haven't used the working solution exactly as 
described by Pierce & Rodriguez but I have used several 
similar ones; all are stable for several years. The 
iron salt can be ferric nitrate or chloride in place 
of ferric ammonium sulphate. (Use an equimolar amount).
There is no need for "stock' and "working" solutions
of eriochrome cyanine R.

I suspect that the two differentiations are not both
needed. The suthors of the J. Histotechnl paper did not 
discuss mechanisms or say how they arrived at their
technique. In general, acids destain everthing except
nuclei, whereas alkali or a ferric salt destains
everything except myelin and erythrocytes. My preference
for a Nissl counterstain would be neutral red, which is
a better contrast to a dark blue myelin stain.

This is a versatile dye that can be used for many
kinds of staining. It can replace haemalum for
nuclei and luxol fast blue for myelin. A mixture
containing dye (orange-red) and its red and blue 
iron-dye complexes at pH 1.5 will impart orange, 
red, pink, purple  and blue colours - similar to 
H&E but quicker, easier and cheaper.

There is plenty of published literature describing 
these methods, which deserve to be used more often. 
Eriochrome cyanine R used commercially as a textile
dye (6 manufacturers listed in C.I. 4th edn) and even 
from lab suppliers it costs only half as much as 
John A. Kiernan
Dept of Anatomy
Univ of Western Ontario
London,  Canada
Inka Tertinegg wrote: 
> Hello,
> Does anyone have a protocol for solochrome cyanine
> stain for GMA sections?
> thanks,
>         Inka

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