Help please Herovici staining quantitation

Hello, I am really new in this imaging processing, and I wonder if
does any one have experience on determining different colors in a staining/
Herovici staining for new and old collagen quantitation. In principle, blue * new collagen( red> old collagen). What is the best way to quantify this images in Fiji?78_4d_W_[13804,37044]_composite_image.tif (2.9 MB)

Please do not keep opening new threads on the same subject.

If you can get a section stained with only the blue dye (pricromethyl blue?) and another with the red (picroacid fuchsin?) then you could generate a 2 colour vector to use with the colour deconvolution plugin, but the issue then becomes how do you compare their intensities. I suspect that this is not a stoichiometric stain, so you would not be able to tell quatitatively how much there is of one or another type of stained material.

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First I sincerely apologise for creating two post with the same problem. I just got loss And couldn’t find the link to my first one. I do have a a staining of each component, but got really lost in translation about the vector suggestion… my idea is to quantify this blue red areas and then validate the results with inmunostaining for specific collagens. It has been challenging…

I suspect that the staining is not really “specify” to collagen “types”.
To detect collagen “types” (I, II, III, etc.) you need a different approach, perhaps immunhistochemistry, but that is not stoichiometric ( either, so while you can tell if a given collagen type is present at a given location, you cannot tell how much there is based on stain intensity (–Lambert_law).

Så, Herovici, was wrong? Or maybe I explained myself poorly. I do not really need to know how much it is. But rather, need to know wether there is and when new collagen fibers are being produced. The complete experiment goes as comparing wound tissue sections over the time… So if the principle of the technique is right, the staining should give a blue staining to newly synthesize collagen fibers, not thighly packaged, and red collagen fibers highly organized.

Is it reliable? See:

As to separating the two colours, you might want to consider colour deconvolution, but please see the warning “note” on IHC, in the link below. I think that the staining method you are using is also non-stoichiometric, so you won’t get a reliable measure of quantity of stained material from the intensity of the dyes.

If on the other hand you just want to use colour deconvolution for segmentation, see the “Determining new vectors” section:

Hello Gabriel,
Thanks for the link and for all the feed back, yes it looks like the staining will also reflect in blue other than new collagens, which also is relevant in this experiment, since I believe that there is a whole complex dynamics of degradation and production of matrix during the wound healing process. I am very grateful for your link with color deconvolution for segmentation. As a new user of imaging analysis, I have a head a lot to study :slight_smile:

Have a nice day,

Hi again,
Would you be able to post the protocol for the stain. The paper does not appear to be available online. Thanks.

Yes the protocol that I used with few modifications was as described by

Neill J. Turner et al, 2017 in J Tissue Eng Regen Med. 2013 Feb;7(2):139-48. doi: 10.1002/term.508. Epub 2011 Nov 9. Quantitative multispectral imaging of Herovici’s polychrome for the assessment of collagen content and tissue remodelling

It is as follow:
Prepare the following solutions:


1% aqueous acid Fucshin ( sigma cat nr 857408)

10 ml deionized water


0.1%Van Gieson staining

495ml picri acid ( satured, sigma)

5m 1% aqueous acid fuchsin


0,05% Methyl blue stain

500 ug methyl blue (sigma cat nr M5528)

100ml 1% acetic acid


50 ml 0.1% van Gieson stain

25 ml 0,05% methyl blue

Use 5 uM deparaffinised and hydrated slides the slides in the polychrome solution for 5 min

  1. remove slide and immidiately place in 1% acteic acid for 1-2 min)

3.Rinse in 100% ethanol for 5 min

dehydrate and clear begining with 100% ethanol ( 1 min) x2

Xylene ( 2 min x 2)

coverslip using non.aqeous montant medium

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Thank you very much.

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