[Histonet] Re: Histonet Digest, Vol 40, Issue 19

[Mark Elliott]

As requested by a few people, here is the method for Hansel's Stain we
have been using.  It was originally modified to work on plastic sections
but it will work on paraffin and cryosections as well.
Mark

Hansel*s Stain

-developed for staining glutaraldehyde fixed, methacrylate embedded
pulmonary tissues.

This technique, developed by Randall J. Thomson and Laura L. Beattie
during research under Dr. R.R. Schellenberg at the UBC Pulmonary
Research Laboratory, is a modification of A.J.P. Scientific*s (1)
method for staining eosinophils in unfixed, air dried urine samples for
the detection of eosinophiluria (2).  The protocol and results reported
are based on guinea pig (Cam-Hartley) airway tissue fixed with
glutaraldehyde (2.5% in 0.1 M Sodium Cacodylate buffer), embedded in
glycol methacrylate (JB-4) plastic and sectioned at 3 um.

Reagents:

The reagents used can be easily prepared in the laboratory, or may be
ordered fresh from A.J.P. Scientific (product # 692).

Solution 1:	0.2% Eosin Y (C.I. 45380) in 100% methanol (3).
(0.1g/50ml)

Solution 2:	1:1 combination of Solution 1 and phosphate buffer (pH
6.8):
		Phosphate buffer, pH 6.8:
			Stock solution a:  9.465g  Na2HPO4/L distilled
water.
			Stock solution b:  9.070g  KH2PO4/L distilled
water.
Use:   49.6 mL stock solution a + 50.4 mL stock solution b to yield 100
mL of buffer.

Solution 3:	0.5% Methylene Blue (C.I. 52015) in 100% methanol (3). 
(0.25g/50ml)
		** for cryosections and cytospins use 0.05-0.005%
methylene blue
** for paraffin use 0.025 g/50 ml or use 0.25g/50ml but use solution 3
for 1 sec, rinse until clear, air dry and coverslip.

Protocol:

1.	Submerge slides in Solution 1 for 60 seconds.
2.	Transfer to Solution 2 for 5 minutes.
3.	Pass slides through two rapid distilled water washes to clear
excess Eosin Y, and allow as much water to drain off as possible before
proceeding to next step.
4.	Submerge slides in Solution 3 for 8 seconds.
5.	Immediately pass slides through two distilled water washes to
clear excess Methylene Blue, and allow slides to air dry IN THE DARK.
6.	Coverslip slides and view.

Results:

Eosinophils stain with pale red cytoplasm and bright red granules,
nuclei stain dark blue, epithelial and smooth muscle cells stain with
medium blue cytoplasm, connective tissue stains with light blue
cytoplasm, mast cell granules stain deep purple, and chondrocyte
cytoplasm and the bulk of the cartilage matrix stain violet.  (Eosin
stains argenine residues in *eosinophilic* granules).

If doing paraffin sections, one can dehydrate to clear as usual, and
then mount with permanent mounting medium.

Warning:

Empirical studies have shown that extended exposure of the slides to an
intense light source (such as that of a projecting microscope) can cause
a photo-reaction leading to a loss of definition of staining (the
mechanism of this effect is unclear), while slides kept in the dark
(while not in use with a standard light microscope) are stable for at
least 5 months (our present maximum storage period).

Notes:

1	A.J.P.  Hansel stain kit insert, A.J.P. Scientific, Inc., PO Box
1589, Union City NJ  Telephone (201) 472-7200.
2	Nolan, C.R., III, M.S. Anger and S.P. Kelleher. 
Eosinophiluria*A new method of detectin and definition of the
clinical spectrum. N. Engl. J. Med.  315:1516-1519, 1986.
3	Use a transparent brown or opaque glass bottle for storage.


Original reference is 
Hansel FK. Clinical Allergy.  St. Louis: CV Mosby, 1953.


Our reference:
Matsuse T, Thomson RJ, Chen X-R, Salari H and RR Schellenberg (1991). 
Capsaicin inhibits airway hyper-responsiveness but not lipoxygenase
activity or eosinophilia after repeated aerosolized antigen in guinea
pigs.  Am. Rev. Respir. Dis.  144:368-372.





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