Toluidine Blue for frozen sections

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From:Becky Scholes <>
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Hi, Histonetters,

Several of you have asked for the Toluidine Blue solution for staining 
frozen sections.  We've discovered that you have to have a dye content of at 
least 85%, so consider that when you purchase the dye.  The publication 
Histologic, Vol XII, No 3, July 1982 had an article that suggested 0.2 to 
1.0 grams of urea added to 50 cc of staining solutions such as Toluidine 
Blue improves the staining quality. We had been using Brig 25 to break the 
surface tension, but found urea worked even better. Maybe it was a fluke, 
but just a warning.  Once our solutions suddenly became extremely pale, and 
after experimenting, we discovered that the
two-year-old dye just....died (OK, I could have used another word, but it is 
6:00 AM - I'm entitled to a pun!)

    urea - 4 gm
    distilled water - 120 cc
    isopropanol (2-propanol) - 280 cc
    Toluidine Blue O - 2.8 gm
   Dissolve urea in water.  Add alcohol.  Stir in Toluidine Blue O.
   Filter.  Shelf life is indefinite.

1.  Tap water to thoroughly rinse off OCT - 35-40 dips
2.  Toluidine Blue - 10-15 dips, depending on the age of the stain.
3.  Isopropanol - 5 changes, 10 dips each.
4.  Isopropanol/xylene - 10 dips
5.  xylene, 2 changes - 10 dips each
6.  Coverslip with permanent mounting media.

Note:  one quick dip in the T Blue on a deparaffinized section is a great 
stain for mast cells!

Becky Scholes
Iowa Pathology Associates
Des Moines, IA
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