RE: [Histonet] How to stain frozen section with Gomori trichrome?
Welcome to the wonderful world of Histonet!
On a frozen section of muscle, this modified Gomori trichrome IS supposed to
have green muscle with slightly lighter green connective tissue.
What is supposed to be red are the mitochondria and the nemaline rods,
which, in normal muscle, you wouldn't see. (On higher power, you might see
very small red dots in the muscle fibers, which are the mitochondria.)
However, in cases of mitochondrial diseases, where there is an increase
number of mitochondria, such as a condition known as red-ragged fibers, then
you will see the accumulated mitochondria. Also, nemaline myopathies would
have an increase in nemaline rods, and these would be seen as red. Plus,
normal nerve fibers will have some red in them, so if you have a section of
larger nerve in the muscle connective tissue, you would see red.
Try going to Google, and right above the rectangle where you type in the key
words, click on "Images" (it's usually defaulted to "Web". Then type in
"Gomori trichrome muscle" (without the ") in the box and hit Search. You
will see lots of images, all green on green, with a few showing the
red-ragged fibers. Or type in "red ragged fiber" or "nemaline rod", if you
want to see the modified Gomori trichrome in these diseases.
Comparison of procedures:
Usual Gomori Trichrome - Uses fixed, processed tissue, with the sections
post-mordanted in Bouins, and the pH of the Gomori Trichrome staining
solution between 1.5-2.5 pH, THEN the muscles are red and the connective
Modified Gomori Trichrome for muscle - Procedure is modified for FS muscle
(tissue is not fixed, not processed, not post-mordanted in Bouins, and the
staining solution is at pH 3.4) specifically so it WILL demonstrate
mitochondria and nemaline rods as red, NOT to differentiate muscle from
A really good site is the Neuromuscular Disease Center from Washington
University in St. Louis, MO.
Hope this helps.
Peggy A. Wenk, HTL(ASCP)SLS
William Beaumont Hospital
Royal Oak, MI 48073
[mailto:firstname.lastname@example.org] On Behalf Of Xilong Li
Sent: Thursday, July 27, 2006 3:39 PM
Subject: [Histonet] How to stain frozen section with Gomori trichrome?
Hi, All members,
I am a new member for histonet. I'm looking for a consistent protocol for a
Gomori trichrome stain for frozen section. I suppose this topic was
discussed previously, but I can not get exact answer when I try to review
early messages. So I send messages here.
I tried to stain frozen muscle section with gomori trichrome but failed
recently. My protocol is as follow in brief:
1 frozen section was brought to room temperature before staining which was
token from -80C.
2 stain nuclei with Weigert's Iron haematoxylin (sigma) for 15min
3 wash in distilled water for 1min
4 stain in staining solution (Chromotrope 2R 0.6g, fast green FCF 0.3g,
Phosphotungstic acid 0.6g, Glacial acetic acid 1.0ml, distilled water 100ml,
adjust pH to 3.4) for 12min
5 rinse in 1%(50ul/50ml) acetic acid in dips
6 dehydration with 100% alcohols in dips
7 put section in xylene 1min
The pictures was so bad without the red color for cytoplasma, almost all
part of muscle tissue or cytoplasma was green or dark(which suppose to be
red color), it was so hard to make clear where is collagen, or nuclei which
was stained in funny red color at some extent. I changed formula of one
step stain solution (Chromotrope 2R 0.6g, fast green FCF 0.1g,
Phosphotungstic acid 0.8g, Glacial acetic acid 1.0ml, distilled water 100ml,
adjust pH to 3.4) according to sigma formula of commercially prepared
solution, and stained again, no improvement was gotten.
So I am just looking forward to hearing from any guys, who can give me some
suggestions what was wrong with my current protocol, and how to improve it,
or just email me the protocol which works in their labs. My email is:
email@example.com, or just fax me 214-648-7902.
Thanks in advance.
Dr. Xilong Li
Hypertension Division, Internal Medicine University of Texas Southwestern
5323 Harry Hiness Blvd-J4.142
Dallas, TX 75390
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