Hale's Colloidal Iron Technique, Culling; 3rd edition

From:Victoria Baker

"Hale's technique is based upon the affinity of acid
groups for colloidal iron at a low pH.  The iron forms
a chelate with the acid groups and may then be
demonstrated by the Prussian Blue reaction.
While this method is excellent for the demonstration
of acid mucins it is inferior to Alcian blue as a
specific method for histochemical investigation.

Special solutions required
Dialysed iron .... 1 volume
2M acetic acid.....1 volume

Dialysed Iron Solution (Reinhardt and Abu'l Haj)
Dissolve gradually 75gm ferric chloride in 250ml
distilled water, adding 100ml glycerol, followed by
the gradual addition of 55ml of 28% ammonia with
constant stirring.  This mixture is dialysed against
regularly changed distilled water for 3 days.

Acid-ferrocyanide Solution
Equal parts of 2% potassium ferrocyanide and 2%HCl.

1.  Bring sections to water.
2.  Flood with dialysed iron solution (either solution
works well)for 10 minutes.
3.  Wash well with distilled water.
4.  Flood with acid ferrocyanide solution for 10
5.  Wash well in water.
6.  Counterstain lightly with 0.1% neutral red or
7.  Rinse in water, and dehydrate rapidly.
8.  Clear in xylol and mount in D.P.X. or H.S.R.


Acid mucopolysaccharides.....Bright blue
Other tissue constituents....Shades of red."

The exact script (minus any typos on my behalf) comes

Handbook of Histopathological and Histochemical
Techniques;  3rd edition, 1974.  C.F.A. Culling. 

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