Re: PTAH stain

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From:"Tony Henwood" <>,
Date:Sun, 29 Aug 1999 20:15:46 +0000
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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 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.


    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.


     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.


     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.


      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

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