Trichrome is the old standard and is a relatively inexpensive method to use. Alfa-smooth muscle actin staining is also very useful to detect myofibroblasts in tissue. You also might want to try CTGF staining as it is selectively induced by TGF-beta and would be a marker of activated myofibroblasts. If you are interested in the later I can get you the method we use in my laboratory.

Dear Patrick Kao - u can refer to http://www.google.com/patents/US6986995.

Many researchers regard interstitial deposition of collagen, stainable by Van Geisson or Sirius Red, as fibrosis.This does not take into account the fact that much of the collagen deposition associated with inflammation is type 3 and this can be a transitory phenomenon. A more classical pathological view is that true fibrosis involves excessive and stable deposition of collagen, commonly type 1, which is cross-linked and stable. You can distinguish between the two with antibodies specific to the various collagen types. A good clue can also be the strong anisotropic qualities of dense type 1 collagen, easily detected by use of crossed polarizers.

On the whole I agree with Terry. The hallmark of fibrosis is increased accumulation of extracellular matrix proteins. Using anitbody based staining is probably the best; one could identify different forms of collagen as well as fibronectin. This tends to be a little expensive for routine or high-through put work. Alternatives are picrosirius red which binds to collagen fibrils and at least in theory allows one to differentiate between collagen types on the basis of differing fibril diameter. Picromallory Trichrome is also quite useful, staining collagen and fibrin in different colours. One would expect to find an increased numbe of myofibroblasts in fibrosis and staining for them using alpha smooth muscle actin provides useful data.

The reply of Mark Docrell includes the usual methods and findings associated with fibrosis. Routine Masson Trichrome is done in most histopathology laboratories. To the methodolgies already enumerated, I would like to add transmission electron microscopy (TEM); although not routine, it will reveal collagen bundles and other fibrils taking part in the establishment of fibrosis, even in early lesions.

Theodore C. Iancu

Technion-Israel Institute of Technology, Haifa, Israel

Hi Patrick,

With immunohistochemsitry or ımmunofluoresans, you can mark anti-alfa-SMA,-collagen I and -collagen III for fibrosis detection. Because the fibrosis is characterized increased ECM deposition (col-I and -III) and alfa-SMA (myofibroblast marker) , and decreased MMPs.

For staining method, you can apply Masson's Trichrome to visualize the fibrosis in tissue. And then, you can do Sircol collagen assay to quantify the collagen content in that tissue.

Trichrome is the old standard and is a relatively inexpensive method to use. Alfa-smooth muscle actin staining is also very useful to detect myofibroblasts in tissue. You also might want to try CTGF staining as it is selectively induced by TGF-beta and would be a marker of activated myofibroblasts. If you are interested in the later I can get you the method we use in my laboratory.

Thank you all for theses precious replies, I guess I'll try trichrome first, to see if my model does have obvious increases of fibrosis. One more thing, does the increase of albumin mean anything in fibrosis? like if the level of salivary albumin increases, can this point to fibrosis within tongue?

Hi Patrick,

Trichrome staining is many times misleading, therefore Picro Sirius red is better. As fibrosis means collagen deposition (usually type I), the easy way out is to stain with Picro Sirius red. You will find the methods and the suppliers for the two reagents on the web. If you cant do staining the solution is to do0 biochemistry with Sircol, that will give you an estimate if ended you are dealing collagen deposition, and this is the first step. After that you can consider if it is worth-it to stain, immunostain or as suggested to do TEM, that by the way will give you more information but with a lot of work. Good luck.

That's an interesting question. and I don't have a definite answer but If you presume that increase salivary albumin is associated with a decrease in oral mucosal integrity I could see that there would be a potential link with sub mucosal fibrosis and I imagine this could include the tongue. That is quite a lot of supposition; I'm not sure you would necessarily have a definite positive association although it is possible.

you can use Masson's trichrome to detect fibrosis with in tissue.

Traditionally fibrosis is detected in tissue by histochemistry using either the Masson Trichrome or the picro-sirius red stains (this will detect collagen fibrils that are deposited in the matrix). You can also check for the presence of myofibroblasts (the major cell type that secretes collagen) through immunohistochemistry by staining for alpha smooth muscle actin. Hydroxyproline, a component of the collagen fibril can be quantitated. Another alternative is immunoblotting for collagen making sure that the antibody is specific for the type of collagen being detected.

Another original way to detect collagen deposition is nonlinear optical microscopy (which is usually called multiphoton microscopy as well). There are two types of signals to be detected: the second harmonic generation (SHG) and multiphoton fluorescence. Please, do a search, it's very popular state-of-art method. You also can find several approaches to quantify the extent of fibrosis.

Just a short remark, it is often required to confirm fibrosis by additional methods in addition to IHC or histology (in which I sprefer IHC and Sirius Red birefringence). Therefore, Western blots in tissue lysates for collagens of fibronectin (or other ECM components), or hydroxyprolin assay or similar (Sircol assay) might be a good way to confirm you results. If you expect small differences between groups use computer based morphometry in IHC, its more sensitive then scoring systems.