Staining, Histochemistry and Histotechnology
(Frequently Asked Questions)
Dr. John A. Kiernan
Department of Anatomy
and Cell Biology
The University of Western Ontario
Can a less toxic oxidizing agent be substituted for mercuric oxide in Harris's alum hematoxylin?
Yes. Mercuric oxide for the oxidation of hematoxylin in Harris's hemalum can be replaced with sodium iodate (NaIO3) or other oxidants:
According to Hansen (1895), one of the following is, in general, needed for the oxidation of 1 gm of hematoxylin to hematein:
* KMnO4: 177 mg
* KClO3: 114
* NaIO3: 197
* KCr2O7: 276 mg
It is advisable to use only half of these quantities, to delay over-oxidation. Vacca (1985) suggested 75 mg NaIO3 per gm hematoxylin, and P. Bock (1989) suggested 98.5 mg NaIO3 per gm hematoxylin.
Bock, P.: Romeis' Mikroskopische Technik; 1989
Hansen, F.C.C.: Eine schnelle Methode zur Herstellung des Bohmersen Hematoxylins. Zoolog. Anz. 473; 1895.
Vacca: Laboratory Manual of Histochemistry; 1985.
Almost every hematoxylin can be used regressively, my favorite for general histology is:
"Mayer's acid hemalum, modified by Lillie":
"Dissolve 5gm hematoxylin by holding overnight in 700 ml distilled water; add 50 gm ammonium alum and 0.25 gm NaIO3. After these have gone into solution, add 300 ml glycerin C.P. and 20 ml glacial acetic acid. May be used immediately; stain for 5 min."
Procedure. (5-7 æm paraffin sections, fixation: Bouin; manual staining.)
* Sections to distilled water.
* Sections to alum-hematoxylin (3 min).
* Sections to acid alcohol (2-3 dips or until
* Rinse sections in tap water (about 10 sec, until most of
the acid alcohol has dissapeared from the slide).
* Rinse sections in 1% NaHCO3 in distilled water (1 min).
* Rinse sections in distilled water (1 min).
* Sections in 0.5% eosin Y in distilled water (30 sec).
* Rinse sections in distilled water (a few dips, until most of the "free" eosin has dissapeared).
* Dehydrate, clear, mount.