RE: [Histonet] Destaining PAP slides

From:"Lee & Peggy Wenk"

I would guess a lot of the autofluorescence would be due to the eosin. Are
you seening a greenish color? Hematoxylin would actually be blocking the
nuclei autofluorescence. So the cytoplasm would be green glowing, with black
holes for nuclei.

Try decolorizing like an H&E.

Remove the coverslip.

Remove the mounting media with 3 changes xylene or substitute, about 5
minutes each.

2 changes absolute alcohol, 1 minute each

2 changes 95% alcohol, 1 minute each

1 change 70% alcohol, 1 minute

Rinse in running water, 1 minute - (running down through alcohol and going
into water should get rid a lot of the light green and some of the eosin)

Place in 1% acetic acid (or if you use a regressive H&E, use the acid rinse
after the hematoxylin). This should remove the hematoxylin. Dip up and down
for 5-10 seconds, up to 1 minute - check with a microscope.

Rinse in water 1 minute.

Place in the bluing agent used in the H&E (ammonia water, lithium carbonate,
etc.). 5-60 seconds. Check with microcope. This should remove the eosin, and
probably the orange G.

Rinse in water 1 minute.

Should be set to go.

If your formulation has Bismarck brown, I don't know how to remove it,
simply because I never understood how or if it was working in the Pap stain.
It's at the wrong pH - it's in a acidic solution, but it needs a basic pH to
bind to tissue. But, since you are going into 
- water (which removes dyes soluble in water)
- alcohol (which removes dyes soluble in alcohol)
- dilute acid (which removes basic/cationic dyes)
- dilute base (which removes acidic/anionic dyes)

I think we have removed just about every variation of dye there is found in
a Pap stain.

Hope that helps.

Peggy A. Wenk, HTL(ASCP)SLS
William Beaumont Hospital
Royal Oak, MI 48073

-----Original Message-----
[] On Behalf Of Angela
Sent: Thursday, February 28, 2008 2:17 PM
Subject: [Histonet] Destaining PAP slides

Good Afternoon,

Does anyone out there have a good protocol for destaining PAP slides?
I'm really trying to remove not only the color, but the fluorescence caused
by the stain itself.



Angela McNabola, MS HT(ASCP)SLS, QIHC
Research Scientist
Ikonisys, Inc.
5 Science Park
New Haven, CT 06511

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