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

[Gudrun Lang]

 Try to shorten the staining time. The fiber-stain (blue) is likely to
overstain the whole section, when incubation time is too long.
We stained 8 min on FFPE sections. I think frozens stain faster overall.
FFPE-staining without mordanting in Bouin rendered the cytoplasma blue. I
don't know, how frozens behave in this manner.

Hope this helps (a little bit)
Gudrun

-----Ursprüngliche Nachricht-----
Von: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] Im Auftrag von Xilong Li
Gesendet: Donnerstag, 27. Juli 2006 21:39
An: histonet <@t> lists.utsouthwestern.edu
Betreff: [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
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 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:
xilong.li <@t> 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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