Re: PTAH stain
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From: | "Tony Henwood" <henwood@mail.one.net.au> |
To: | Histonet@pathology.swmed.edu, RHD101@aol.com |
Reply-To: | |
Date: | Sun, 29 Aug 1999 20:15:46 +0000 |
Content-Type: | text/plain; charset=US-ASCII |
Dear John,
> Does the PTAH stain have chloroform as one of its ingredients and, if so, is
> there an alternative to the use of chloroform? Are other labs refusing to do
> this stain because of the chloroform and its association with liver damage?
The following technique does not use chloroform. Possibly someone may
have recommended chloroform rather than xylene (or another
substitute) in processing.
CHERUKIAN'S MODIFIED PHOSPHOTUNGSTIC ACID HAEMATOXYLIN
NOTES:
Cherukian's modification employs an eosin solution which stains the
erythrocytes red and differentiates them from the blue fibrin.
FIXATION: 10% buffered formalin.
MICROTOMY: paraffin sections at 5um.
SOLUTIONS:
1. Eosin:
Eosin Y, water soluble (CI 145380) 0.5g
Distilled Water 10ml
80% Ethanol 190ml
Working: 10ml stock and
Before use add 50ul glacial acetic acid.
2. 5% Periodic Acid
3. PTAH solution
Haematoxylin (CI 75290) 0.5g
Phosphotungstic Acid 10g
Distilled water 500ml
Dissolve solid ingredients in separate portions of the
water. Use gentle heat for Haematoxylin. Combine solutions
when cool. Add 0.088g potassium permanganate to ripen. The
stain is ready to use.
CONTROLS:
Use brain sections and section of muscle. A good stain will
demonstrate the dendrites as blue where as in a bad stain they
appear light grey to salmon in colour. Nuclei, fibrin, platelets
and muscle will be blue, red cells and collagen appear red.
Muscle striations should be well defined.
STAINING PROCEDURE:
1. Dewax and hydrate sections to 80% alcohol.
2. Place slides in eosin for 30 seconds.
3. Wash slides in distilled water for a few seconds.
4. Place slides in 5% periodic acid for 10 minutes.
5. Wash slides in water for 3 minutes.
6. Place slides in PTAH for 30-90 minutes in 60 oC oven.
Check from 30 minutes on.
7. Dehydrate, clear and mount.
REFERENCES:
Cherukian, C.J., Histologic. 8(4); 105, (1977).
Luna, L., Histologic. 5(2); 66, (1975).
.
Tony Henwood
Senior Scientist
Anatomical Pathology
Royal Prince Alfred Hospital
Sydney, AUSTRALIA
http://www2.one.net.au/~henwood
http://www.pathsearch.com/homepages/TonyHenwood/default.html
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