Re: [Histonet] frozen section trichrome

From:John Kiernan

Expect funny results when trichrome stains (devised
for paraffin sections) are done on frozen sections.
There have been a number of recent Histonet postings
on this subject. See and search
for "trichrome frozen" (without the quotation marks).

In 1963 WK Engel & GG Cunningham (Neurology 13: 919-923)
published a modified trichrome for cryostat sections of
unfixed muscle (human biopsies and a wide variety of
animals). It differs in important ways from its parent
method (Gomori's one-step trichrome). My comments on
the stages of the technique are indented. (I hope it
looks OK after it's been emailed. If it's a real mess,
please let me know.)

There is no pre-staining treatment with picric acid
or Bouin's fluid, as is usual before doing a regular
trichrome on paraffin sections of formaldehyde-fixed

1. Unfixed sections stained 5 min in Harris's haemalum.
      In a sense, the tissue is also being fixed at
      this stage, because the stain contains a large
      excess of aluminium ions over haematein molecules,
      and it is quite acidic (pH about 2?). 
         A solution of an aluminium salt doesn't 
      work as a fixative for pieces of tissue 
      (see Histochem. J. 17:1131-1146, 1986) but
      aluminium salts do make thin layers of gelatin 
      insoluble in warm water, and are used as hardening 
      agents for photographic films and printing papers.  
2. Wash in distilled water X3.
      Note there was no acid-alcohol differentiation
      of the haemalum, and no blueing step with the
      washing. (There would be no point because the
      next step has pH less than 5, which can be
      expected to change the haemalum colour from blue
      to a dull, weary red.) 
3. Stain for 10 min in Gomori's one-step trichrome 
   solution that has been adjusted to pH 3.4 by adding 
   1N (4%) NaOH.
      For paraffin sections, there's published work to
      support lowering the pH of Gomori's mixture, to
      enhance the contrast between cytoplasm and thin
      collagen fibres. The anions of phosphotungstic
      acid (PTA) change into other anions (some larger 
      and some smaller than PTA) in solutions with pH
      above 2. In most trichrome methods the pH of the
      solution containing PTA (or PMA) is less than 2.
         Engel & Cunningham's 1963 paper contains no 
      explanation of the experiments that led them to
      pre-stain in Harris's haemalum, and there is no
      description of staining by Gomori-type trichrome
      solutions at pH other than 3.4. 
4. A few dips in 0.2% acetic acid.
      The authors wrote "differentiate by a few dips 
      in 0.2% acetic acid." Acidified water PREVENTS
      removal of anionic dyes, and is the opposite of
      differentiation in this technique. Engel &
      Cunningham (1963) did not advise checking wet 
      slides under a microscope at any stage.
5. "Dehydrate and mount in Permount."
      The authors didn't give details. For trichrome
      methods I find it best to go straight from
      the acidified water into the first of three
      changes of 100% alcohol. (Alcohol-water
      mixtures can extract some of the bound dyes.)
Unfortunately Engel & Cunningham (1963) did not 
give any account of the experiments that led to the
development of their procedure. Neither did they
discuss the possible mechanism of action, other than
showing that the red staining of intermyofibrillar
cytoplasm was prevented by strong lipid solvents, and
therefore probably depended on intact mitochondria
and sarcoplasmic reticulum. At pH 3.4 and preceded
by 5 min in Harris's haemalum, the staining mechanisms
must differ from those of regular trichromes, which 
are still controversial. See Prento 2001 Biotech.
Histochem. 76:137-161 for some fairly recent 
discussion of the suject.

John Kiernan
Anatomy, UWO
London, Canada.
Steven Coakley wrote:
> I'm looking for a consistant protocol for a trichrome stain for FS.  I tried 2 this morning on liver.  Both were sectioned at 7u , air dried for 30 minutes, fixed in bouins for 30 minutes and stained.
> Extreamly red with a granular appearence.  Only green was in the folds.  My paraffin section control was fine.
> Steve
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