Picro sirus red staining- troubleshooting

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nandu
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Picro sirus red staining- troubleshooting

Dear all

"Times New Roman","serif"">Yesterday I did picro sirus red staining for the first time to detect Collagen on paraffin embedded tumor samples. I have followed the exact protocol mentioned in IHC world.  It looked fine, with red colored collagen fibers till I added DAKO fluorescent mounting medium.  As soon as I added mounting medium, the red color has leaked out retaining only the nuclear staining.

"Times New Roman","serif"">Here is the protocol I followed.
1. De-wax and hydrate paraffin sections.
2. Stain nuclei with Weigert's haematoxylin for 8 minutes, and then wash the slides for 10 minutes in running tap water).
3. Stain in picro-sirius red for one hour
4. Wash in two changes of acidified water.
5. Physically remove most of the water from the slides by vigorous shaking.
5. Dehydrate in  100% ethanol for 5 minutes
6. Clear in xylene and mount (around 5 minutes)
7. Mounted with DAKO fluorescent mounting medium.
 
Within half an hour almost all the red color has leaked out!
 
Please help.
 
Thank you

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Arvind Singh Pundir
Arvind Singh Pundir's picture
 Dear nandu try changing the

 Dear nandu try changing the mounting media i dont know why you have used Fl mounting media

nandu
nandu's picture
thank you pundir

thank you pundir

We have only flourescent mounting medium in the lab so i have used it. I think you are right. I should change it. Thank you! Good luck with your work.

Arvind Singh Pundir
Arvind Singh Pundir's picture
 Dear Nandu 

 Dear Nandu 
you should use some Organic mounting media as the Xylene is used in your procedure which is a orgaic and interferes with your mounting media may bleached it

nandu
nandu's picture
Thank you!

Thank you!
I will use xylene based mounting media.
Best