[Histonet] Picrosirius red stain
Jeffrey Wu
wurochi <@t> hotmail.com
Tue May 10 12:30:54 CDT 2005
Hello all,
I am trying to quantitate the amount of fibrosis in mouse cardiac
tissue. I based my method on Dr Kiernan's excellent tutorial on
picrosirius red staining (posted in 2000). Briefly, slides are
deparaffinated in xylene (2x, 6 min); 100% ethanol (2x, 3 min); 75%
ethanol (1x, 3 min); and running distilled water (10-15 min). Next,
they are stained in saturated picrosirius red for 60 minutes and
washed in acetic acid (2x, 3 min). They are dehydrated in 75% ethanol
(1x, 3 min); 100% ethanol (2x, 3 min); and xylene (1x, 3 min).
Finally, the slides are mounted with Permount.
For image processing, I am using circularly polarized light. Under
the microscope, the collagen stains from pink to dark red (almost
black), depending on its content; however, the background tissue
appears green/blue. It is problematic because I quantify the
collagen using ImagePro Plus software, in which I select certain
colors. Especially with the pink and some dark blues, there is
overlap between the collagen and tissue, causing overestimation of
collagen. (Sorry for the long explanation.) I guess what I am
seeking is the flaw in my procedure, causing the other tissue not to
stain yellow.
On a side note, I am using the correct picrosirius red stain, and the
solution is saturated with crystals at the bottom. I have
tried modifying washing times without any change. I used a short (2
min) 0.2% phosphomolybdic acid wash, which is supposed to reduce
background, with no success as well.
Once again, sorry for the long question. Any help with my problem
would be greatly appreciated. Please do not hesitate to email me with
any questions or anything that I have not made clear. Thank you in
advance.
J Wu
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