Hi Florent,

looking at your pictures, the second analysis is much closer to what I would expect than the first one. If you are in doubt which of the staining is really collagen, looking at your PSR-stained slides using polarized light microscopy might help. Collagen bundles will light up brightly. In our hand imageanalysis in these kind of slides with ImageJ works best by making a RGB-stack and threshold the green slice using the Yen method. It looks like your pictures are slightly underexposed which makes things harder as well.

good luck,

Reinout

Hi Florent,

I can endorse what Reinout has written above . Though the second Analysis looks bit promising still not so accurate with the marked area. Here we use either Definiens or Histoquant for such morphometric Analysis.

Good luck !

Kanishka

Here is my "no cost" protocol (it is a bit old but and you will have to adjust for FIJI and where an a is below think of an arrow)

In bright-field microscopy collagen is red on a pale yellow background. (Nuclei, if stained, are ideally black but may often be grey or brown. The long time in picro-sirius red causes appreciable de-staining of the nuclei. This is not a problem with traditional van Gieson or with picro-aniline blue, with their 1-minute staining times.)

When examined through crossed polars the larger collagen fibers are bright yellow or orange, and the thinner ones, including reticular fibers, are green.  According to Junqueira et al. (1979) the birefringence is highly specific for collagen. A few materials, including Type 4 collagen in basement membranes, keratohyaline granules and some types of mucus, are stained red but are not birefringent. It is necessary to rotate the slide in order to see all the fibres, because in any single orientation the birefringence of some fibres will be extinguished. This minor inconvenience can be circumvented by equipping the microscope for use with circularly rather than plane polarized light (Whittaker et al., 1994; Whittaker, 1995), but then you don't get a completely black background.

Collagen content was assessed both by morphometric analysis of Sirius red staining of liver sections. For this purpose paraffin embedded sections were deparaffinized and then incubated in Sirius Red solution (saturated picric acid containing 0.1% Direct Red 80 and 0.1% Fast Green FCF) for 2 hours. After that, section were dipped in distilled water shortly and then washed with 100% ethanol.

Sections were kept in xylene until mounting. The area of positive Sirius red staining was measured using IPLabTM software. At least five low power fields were analyzed for each mouse.

Converting Image to Appropriate Greyscale image for Image J Interpretation

1) Adjust levels à enhance to adjust brightness contrast to levels.  Try not to make too bright or too dark.

2) Enhance RGB (white out background and darken red as much as possible)

3) Image à mode à greyscale

4) Resize Image to 500 pixel width

Interpretation using Image J Software

Plugins à Macros à Install à Measure Area Fractions

File à Open à Open File

Image à Adjust à Threshold (you can set your own threshold or use auto)

Process à Binary à Make Binary à opens a dialog box about Thresholds à just press OK

Plugins à Macros à Measure Percent Area

thanks all for your response! If I understand well, I have to check first the slide with polarized light microscopy to have a better idea of what is fibrosis and what is not.

Kanishka Hattatiya: maybe I will consider one of these softwares if I really can't use the free ImageJ.  If I use one of these softwares, I will contact you for more details. Thanks a lot!

George Notas: Thank you! I will try this method. 

Hi Florent,

Why not use a chemical assay instead, and measure L-hydroxyproline content in your tissue samples? This offers the advantage of an absolute quantitative result instead of measurements whose values are conditional on your software's settings. Liver works extremely well because little collagen is present in a healthy organ.

http://www.ncbi.nlm.nih.gov/pubmed/21462164

Hope this might be a useful alternative, and good luck,

Luke

Luke Barron: thank you.  It could be a good alternative to the sirius red staining. I will take into consideration.

Hello.

We are working in a bile duct ligature model with image analysis for collagen quantification using ImageJ.

This is the M&M of our next publication:

"Ten non-overlapping photographs from random fields per section were taken from two slides per specimen at 100x magnification. Images were exported in .JPG format with a resolution of 1200 x 900 pixels in RGB color palette. Images were processed in an automated image analyzer (ImageJ version 1.48, U. S. National Institutes of Health, Bethesda, MA, USA). Threshold colors were adjusted using the Hue-Saturation-Brightness (HSB) combination to select all the positive Picrosirius marking according to controls. Selected area was measured in square pixels and the staining intensity was measured in an 8-bit gray scale (0 to 256). The positive area fraction considering a complete picture of the same resolution was calculated. The process of analysis was automated using macrocode to process 10 images per cycle. Data were expressed as a percentage of total liver tissue area."

Puedo responder conseguir algunos parámetros para ayudarte más y ponerte en contacto con quien nos ayudó.

Matias Garrido. Thank you. I will contact you by private message.