RE: How do I know which version of a protocol to use for the HTL Practical?

From:Rena Fail

  In both versions a "red" counterstain is used after the Prussian blue
reaction has taken place. Most versions do use a red counterstain, but
that choice is ultimately yours. You may want to invest a little time in
researching the effects of harsh decalcification on iron staining. Good
Rena Fail
Medical University of SC

-----Original Message-----
From: Miriam Schroeder (by way of Histonet) [] 
Sent: Friday, March 07, 2003 11:26 PM
Subject: How do I know which version of a protocol to use for the HTL

Hello everyone.  I am preparing slides for the HTL practical and am
someone out there might
have some familiarity with how the slides are graded.  In particular, I
need to know how to figure
out WHICH version of a stain is THE "official" version.

For example:  I am requested to do an Iron stain on Bone which includes
marrow.  The extent of my
instructions is this: "Iron (Prussian Blue Reaction)".

In our lab, for an iron stain, we typically follow the method listed on
page 179 of the 3rd
edition of the AFIP.  This stain is called "Gomori's Method for Iron".
uses Hydrochloric Acid,
Potassium Ferrocyanide, and NUCLEAR fast red.  (And, because we have had
bad luck with Nuclear
fast red, our pathologist requests a Hematoxylin counterstain instead.)

Since that stain is not specifically called "Prussian Blue", I've been
looking the
3rd edition of "Theory & Practice of Histological Techniques" (by
& Stevens) there is a
protocol "Perls' Prussian Blue reaction for ferric iron [Perls, 1867]".
This protocol calls for
Potassium Ferrocyanide, Hydrochloric Acid, and NEUTRAL red.  Can I
that becuase of the
specific name "Prussian Blue" that this is the proper protocol I should
follow?  How can I know
(in this sort of situation in general) WHICH version of a protocol the
will want me to use
for my slides?  I have a number of books I consider to be histology
"bibles" but their protocols
often vary on little details like this.  Or will they not be so strict
about these aspects (like
the counterstain), as long as the Prussian Blue part is correct?  How
can I
be sure that a
counterstain is expected at all?  Is there a "histology bible" floating
around out there somewhere
that "trumps" all others?

Any advice that can be given would be appreciated.

Also - does anyone know of a website where I could view images of what
most "official"
versions of various stains should look like?

Thanks in advance,
Miriam Schroeder
Research Associate, Berlex Biosciences

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