Fellow HistoNet Followers-

I recently made mention of the above procedure without providing a reference
or other information.  Several people have asked me for specifics, which I
am now happy to provide.  The following method was developed from a
procedure found in Lillie, R.D. Histopathologic Technique and Practical
Histochemistry, 3rd. ed. 1965.  The Cresyl-echt violet is a nice touch as a
counterstain, but not needed.  It is not part of the original method.

Fixation:  10% NBF
Control:  Medulla

METHOD:
	1)  Deparaffinize and bring sections to water.
	2)  Stain in preheated hematoxylin solution in 60 degree C
oven:.................1 hour
	3)  Differentiate in sodium borate / potassium ferricyanide
solution:............	2-3 min.
	4)  Rinse in deionized water.
	Check slides microscopically.  Nuclei should be decolorized, but red
corpuscles are brown to 	black, and myelin sheaths are black.
	5)  Wash in running deionized
water:........................................................5 min.

	6)  Stain in preheated cresyl-echt violet in 60 degree C
oven.......................5 min.
	7)  Differentiate in two changes of 95%
ethanol:....................................20 sec. each
	8)  Dehydrate in 2 changes absolute ethanol (denatured or
not).................1 min. each
	9)  Clear (xylene or substitute).
	10) Coverslip in synthetic mounting medium.




SOLUTIONS:

Iron Hematoxylin ( DO NOT USE WEIGERT's):
	A.	Ferric
Chloride...................................................2.0 g
		Ammonium
Chloride...........................................0.6 g
		Combine.  Add deionized water to make 100 ml.  Check pH
(should be ~2.0).

	B.
Hematoxylin......................................................1.0 g
		95%
ethanol.....................................................100 ml

		Mix equal parts of A and B.  Place in a 60 degree C oven to
preheat.
		Solution is not stable.  Discard after use.

0.1% Cresyl-Echt Violet Solution (AFIP, 3rd ed., p. 204):
	Cresyl Echt
Violet..........................................................0.1 g
	Deionized
water............................................................100 ml
	Combine and heat to 60 degrees C.
	Just before use, filter and add 15 drops of 10% glacial acetic acid.
	Discard after 3 slide carriers.

Differentiator:
	Sodium borate
(borax)....................................................0.5 g
	Potassium ferricyanide (K3Fe(CN)6)..............................1.25
g
	Deionized
water............................................................100 ml
	Discard after 2 slide carriers.

10% Glacial Acetic Acid:
	Glacial acetic
acid..........................................................10 ml
	Deionized
water..............................................................90 ml
	Expiration date: 1 year.


RESULTS:

This stain has been particularly useful in demonstrating degenerative
changes in the brain following treatment with neurotoxins.
		
BLACK:		Myelin
		Eosinophil Granules of anterior pituitary
		Eosinophil Granules of eosinophil leukocyte

VIOLET:	Nissl 



I hope this helps.

Joe

Joseph A. Saby, BA, HT(ASCP)
Drug Safety Evaluation
Pfizer Global Research and Development
2800 Plymouth Road
Ann Arbor, MI 48105
Phone: (734)-622-3631
FAX:   (734)-622-3866
E-mail: joseph.saby@pfizer.com