Re: Hales

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From:Tony Henwood <>,
Date:Mon, 12 Apr 1999 22:36:28 +0000
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Try the following colloidal iron stain. It works quite well.



At low pH, colloidal iron is absorbed onto tissue polyanions, 
which is then visualised by conversion to ferric ferroycanide 
(Prussian Blue) using Perls technique. This technique is usually 
used on skin biopsies where one is aiming to demonstrate 
Granuloma anularae (loss of connective tissue mucin).

FIXATION:      10% buffered formalin (avoid Dichromate fixation).

MICROTOMY:     4um paraffin sections.


1.   29% ferric chloride solution.

2.   Colloidal iron stock solution:

     Boil 250ml distilled water.  While still boiling, add 4.4ml 29% ferric 
     chloride and stir.  Continue boiling for 5-10 minutes (solution should 
     be dark red).

3.   12% acetic acid (2M).

4.   2% potassium ferrocyanide.

5.   2% HCl.

6.   Colloidal iron working solution:

     Colloidal iron stock solution                20ml
     Distilled water                              15ml
     Glacial acetic acid                           5ml
     Mix just before use.

7.   Kernechtrott.


1.   Stomach.

2.   A second slide should be left in water until step 6 (to 
     detect endogenous iron)

3.   Alcian Blue (pH 2.5) can be run in parallel.


1.   Dewax and hydrate sections.

2.   Place in 12% acetic acid, 5 minutes.

3.   Treat with working solution, 1 hour.

4.   Wash in 12% acetic acid, 5 minutes.

5.   Wash in tap water.

6.   Stain in equal quanitities of 2% K. Ferrocyanide and 2% HCl, 
     5 minutes.

7.   Wash in water.

8.   Counterstain in Kernechtrott, 3 minutes.

9.   Wash, dehydrate, clear and mount.

RESULTS:       Carboxylated Mucins     blue


AFIP manual of Histopathological Techniques, p 167-168.

Bancroft and Stevens, "Theory and Practice of Histopathological 
Techniques", p. 155.
Tony Henwood

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