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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