Re: [Histonet] Perl's Prussian blue Iron stain

From:Paul Bradbury

Hi Ryan,

A couple of comments regarding iron "staining". First, the demonstration 
of iron using the Perls' procedure is not a staining technique. It is a 
histochemical reaction between the ferric ions and potassium ferro 
cyanide, the visible end product is ferric ferro cyanide (also known as 
Prussian blue). This end product is permanent and will not fade under 
normal conditions.

Iron in any form in blocks and sections is remarkably stable. You can 
store sections for years, or you can stain them immediately after drying 
following cutting.

Nuclear fast red is an inherently pale nuclear stain. It is popular as a 
counterstain in the Perls' procedure because it is so unlikely to mask 
any very fine iron reactions. Some of the other red nuclear stain, such 
as neutral red or saffranin, are more potent and will produce much more 
non-nuclear coloration unless they are washed throughly after staining.

If your control is liver, you may anticipate pale reactions all round. 
Liver generally, unless it is from a case involving an iron-storage 
disease, has very little demonstrable iron. Also, the nuclei of 
hepatocytes stain very pale as their chromatin is very diffuse.

Lastly, regarding your staining procedure. I would suggest the following:

    Xylene - 2 changes - 3 minutes each
    100% Alcohol - 3 changes - 1 minute each
    95% alcohol - 1 change - 1 minute
    Distilled water - 2 changes - 1 minutes each (to remove any 
contaminant iron from other sources)
    Ferrocyanide/HCl working solution - 20 minutes
    Distilled water - 2 changes - 1 minute each
    Continue with the other steps that you currently use, they are just 

Paul Bradbury,
Kamloops, Canada

Ryan Dominique Salazar wrote:
> Hi everyone,
>   Please help me on Iron staining. These are my questions:
>   -do I need to stain quickly after heating in the hot plate?
>   -very pale nuclear fast red (Sigma) stain.
>   -Is this OK for my working solution?
>   Hydrochloric Acid-Potassium Ferrocyanide Solution
>   2% hydrochloric acid --------------------------------------------        20.0 ml
>   1% potassium ferrocyanide -------------------------------------          20.0 ml
>         Prepare just before use and discard after use.
>   -my positive control is liver, but it seems the stain is pale, do I need to change my procedure?
>                  3 changes of xylene (3 min each)
>                  3 changes of abs. ETOH (3 min @)
>                  70% ETOH
>                  80% ETOH
>                  HCl-K Ferrocyanide Working solution (30 min)
>                  5 changes of distilled water
>                  Nuclear fast red (3 min)
>                  3 changes of distilled water
>                  70% ETOH 3 dips
>                  80% ETOH 3 dips
>                  3 changes of Abs. ETOH (3 dips @)
>                  3 changes of xylene (3 dips each)
>                  Mount with entellan
>   -Please provide for me any procedures/techniques
>   Thanks fo your big help. Your advice regarding the brain tissue section works well.
> ---------------------------------
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