[Histonet] Alternatives to Weigert's Iron Hematoxylin for Mucin andTrichrom e Staining

Fri Aug 8 20:11:00 CDT 2008

One of the problems with traditional Weigert Hematoxylin is that the ferric
chloride (mordant) will also overoxidize the hematoxylin in 1-3 days, so it
has to be made up every couple of days. This wastes our precious
hematoxylin.

Back in 1997, Histonet ran a Ferrous Hematoxylin, made with sodium iodate as
the oxidizer, and ferrous sulfate and aluminum chloride as the mordants (and
powdered hematoxylin, of course). Make it up, allow to oxidize 3 days at
room temperature. Then store in the refrig. Reuse over and over until weak.
One of my students tested this out, and used it every day, and it finally
started to go weak about 3 months later. Make it on Friday, ready for use on
Monday.

This would be a good was to save on amount of hematoxylin.

(Sorry Jim Elsam (who originally posted it) and Pete Jackson (whom Jim
credits with giving him the procedure), but in our student lab, we call it
Eva's hematoxylin, after our student who tested it out.)

FERROUS HEMATOXYLIN
(Procedure written by Beaumont Hospital, Royal Oak, MI)

PURPOSE:
This stain demonstrates nuclei. When used with Trichrome stains, it
withstands decolorization of the nuclear staining longer than Weigert
Hematoxylin.

PRINCIPLE:
The mordants are ferrous sulfate and aluminum chloride. The oxidizer is
sodium iodate. Since this is an iron hematoxylin, the nuclei are black.
However, since ferrous sulfate is a poor oxidizer, sodium iodate is used as
an oxidizer. The sodium iodate slowly oxidizes the ferrous hematoxylin to
ferrous hematein, so it must sit for about 3 days before use. However, it
does not rapidly over-oxidize to ferrous oxy-hematein, so the solution will
continue to stain nuclei black for about 2 months or longer. Hydrochloric
acid is used to control the pH.

FIXATION:
Any well fixed tissue.

TECHNIQUE:
Cut paraffin sections at 5 um.

CONTROL:
Use the control for which the special stain is needed.

QUALITY CONTROL:
1.	Allow the solution to ripen for 3 days before use.
2.	Store in the refrigerator, to slow down the rate of over-oxidation.

CAUTION:
Follow standard safety procedures when preparing stains.

HEMATOXYLIN is incompatible with oxidizers and alkalies. Store separate from
these.
ALUMINUM CHLORIDE, HEXAHYDRATE is an irritant. Moisture sensitive, as
hydrochloric acid could be created with the addition of water.
FERROUS SULFATE, HEPTAHYDRATE is an irritant. Store dry chemical in
refrigerator. Ingestion of high amounts can cause GI disturbances, liver
damage, and even death.
SODIUM IODATE is an oxidizer. Store away from other material.
HYDROCHLORIC ACID is an acid. Add slowly, drop by drop, to solution. May
cause severe skin and eye burns.

REAGENTS:

SOLUTION A - Ferrous Hematoxylin
Hematoxylin (CI 75290)		0.5 g
95% Alcohol, reagent		50.0 mL
Dissolve together. Store at room temperature. Stable for several months.

SOLUTION B - Ferrous Hematoxylin
Aluminum chloride, hexahydrate (AlCl3.6H2O)		5.0 g
Ferrous sulfate, heptahydrate (FeSO4.7H2O)		5.0 g
Distilled water		                              50.0 mL
Dissolve together. Store at room temperature. Stable for 2 months.

SOLUTION C - Ferrous Hematoxylin
Sodium iodate (NaIO3)		0.9 g
Distilled water		     10.0 mL
Dissolve together. Store at room temperature. Stable for several months.

WORKING SOLUTION - FERROUS HEMATOXYLIN
Solution A		25.0 mL
Solution B		25.0 mL
Mix together. Then add:
Hydrochloric acid, concentrated (HCl)	0.5 mL
Mix together. Then add:
Solution C		0.5 mL
Stir together. Allow to ripen for at room temperature a minimum of three
days before use. Store in refrigerator (3o C.). May be reused until weak.
Stable for 2-3 months, if used every day. May last longer if used less
often. If use this stain for more than one coplin jar at a time, double the
amount.

PROCEDURE:
1.	Use in place of Weigert Hematoxylin in any staining solution, for
same amount of time.

RESULTS:
Nuclei		black

PROCEDURAL NOTES:
1.	May be used cold. Usually do not need to extend time.
2.	Allow to oxidize (ripen) for a minimum of three (3) days before
using. Unoxidized (new) Ferrous Hematoxylin will not stain nuclei.
3.    Store in refrigerator after it has ripened (Note #2), to extend life
of stain.
4.	Over-oxidized (old) Ferrous Hematoxylin shows pale gray-black to no
staining. Throw out solution.

REFERENCES:
* Jim Elsam email to Histonet, October 6, 1997. He credits Pete Jackson at
Leeds, England.

Stain investigated by Eva Odish, HTL(ASCP), when she was a student in the
HTL program at Beaumont Hospital, Royal Oak, MI in 2000.

* Source of procedure

Peggy A. Wenk, HTL(ASCP)SLS
Beaumont Hospital
Royal Oak, MI 48073

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of McKnight,
Tanisha
Sent: Friday, August 08, 2008 1:26 PM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Alternatives to Weigert's Iron Hematoxylin for Mucin
andTrichrom e Staining
Importance: High

 Hello All:

Can anyone suggest an alternative to Weigert's Iron Hematoxylin for nuclei
staining with Mucin and Trichrome protocols? I have some standard
hematoxylin (7211) that I will be using for my routine staining. However,
with the shortage, Weigerts is on backorder until October. 

Tanisha McKnight, HT(ASCP)
AP-Histology/Specimen Management
Covance CLS, Indianapolis

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