[Histonet] How to stain frozen section with Gomori trichrome?

From:"Xilong Li"

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 
8 mount

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

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: xilong.li@utsouthwestern.edu, or just fax me 214-648-7902.

Thanks in advance.

Dr. Xilong Li
Hypertension Division, Internal Medicine
University of Texas Southwestern Medical Center 
5323 Harry Hiness Blvd-J4.142
Dallas, TX 75390

Tel: 214-648-9966(L)
Fax: 214-648-7902

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