[Histonet] Quantification for Sirius red staining

From:Jamie E Erickson

Hi Amy,
                 I have done this using polarized light with Image pro 
plus.  Also here is a brief procedure of what I did, I was trying to 
quantitate collagen in mouse disease lungs. You should remember if you are 
trying to quantitate this let your scope warm up for 30 minutes prior to 
capturing your images and keep all light settings (levels) / image size 
the same. Your lamp temperature as it warms can give you different reading 
(IOD) if not warmed up. I used a polarizer that screwed into to my scope 
so I could rotate it to the right degree (I think 45) and it sits in place 
so as not to move. I also did not use any NDF filters and I used the mouse 
Kidney as positive control. 

Here is a reference that I followed.

1. Gaoyun Yan,  2005 Therapeutic Dosing of anti-IL-13 Monoclonal Antibody 
inhibits Asthma Progression in Mice. Journal of Pharmacology and 
Experimental Therapeutics. 313: 8-15.
2. Juqueira LC, Picrosirius staining plus polarization microscopy, a 
specific method for collagen detection in tissue sections. J. 
Histochemistry 11(4) 447-55, 1979. 
3. Histonet search: 

picrosirius red (PSR) (EMS, Cat #26357) for Collagen deposition.

Picosirious Red Imaging

Slides where stained, imaged and saved as stated above. Images for PSR 
collagen deposition where photographed under polarized light microscopy, 
which yields a yellow, red and green birefringence of collagens type I, 
II, III (Appendix 3). Background non-specific birefringence is removed as 
stated above. Images are converted from RGB color images to HSI images and 
the Intensity channel is used for analysis. The images are calibrated and 
Threshholded on the red and yellow collagen fibers which represent mostly 
the type I fibers. These areas are measured and counted using the 
integrated optical density (IOD) feature, which represents the area 
multiplied by the average intensity. This measurement is then exported to 
excel and the two images for each sample are averaged to arrive at the 
average intensity for the total sample. 

Hope it helps.


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