Hi Davoud,

On ImageJ, first step should be to separate the RGB channels and select the one where the collagen is (blue for trichrome staining or red if you used Sirius red) (Image>Colour>Split channels). Then, you have to make a threshold to select only the collagen signal. This depends substantially on the quality of your images. If they have a lot of noise, try to do a manual B/C adjustment first (Image>Adjust>Brightness and Contrast) and then the threshold (Image>Adjust>Threshold) to select only the collagen as black and the rest as white. Go to Analyse>Analyse particles and get the area for each collagen region. You can finally sum the values and get the amount of collagen on your picture.

Hope this can be helpful! Best regards.

Thank you for your response dear Mohamed Mahdy and Daniel Carvalho . Do I have to stain the cutaneous sections with specific collagen stain ( Trichrome or Sirius Red ) or can I perform the procedure on Hematoxylin and eosin stained specimens which I have in my disposal?

Hi Davoud,

Yes, you have to use Picrosirius Red or Trichrome staining. We use picrosirius red specifically for collagen staining.

Daniel measurement protocol works very good; but, you could try using any macro for quantification available for ImageJ/FIJI, like the one by Awal M. Hadi et al. in:Article Rapid Quantification of Myocardial Fibrosis: A New Macro-Bas...

Macros are very helpful when you have many sections and/or pictures per section.

Good luck!

If you use picrosirius red for collagen staining is possible (using a polarized light microscope) to quantify the collagen. The collagen is birefringent in polarized light. It "looks" exactly like as a probe in fluorescent light. In this way the next steps in analyzing with ImageJ is more easy.

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