The reason for low immunofluorescence signals of collagens is not their poor immunogenicity , no matter how correct it is that collagens are poor immunogens. The reason is that 1) collagen I fibres do not exist. All collagen fibrils or fibres are macromolecular alloys comprising several collagens and also non-collagenous macromolecules. Do not think of a fibre as a heap of collagen molecules (which is what most people do). 2) The latter are enriched in the periphery of the fibres and mask the epitopes of most anti-collagen antibodies. Unmasking is required to obtain good immunofluorescence signals. In some cases, treatment with acid or heat is not sufficient to expose the epitopes but partial degradation by proteolytic enzymes helps in those cases.  3) The exact treatment depends on the tissues and collagens investigated. Literature: Hendrix, M.J.C., E.D. Hay, K. von der Mark, and T.F. Linsenmayer. 1982. Immunohistochemical localization of collagen types I and II in the developing chick cornea and tibia by electron microscopy. Invest. Ophthalmol. Vis. Sci. 22:359-377; Mendler, M., S.G. Eich-Bender, L. Vaughan, K.H. Winterhalter, and P. Bruckner. 1989. Cartilage contains mixed fibrils of collagen types II, IX, and XI. J. Cell Biol. 108:191-197; von der Mark K, von der Mark H, Timpl R, and Trelstad RL: Immunofluorescent localization of collagen types I, II, and III in the embryonic chick eye. Dev Biol 59:75, 1977. Incidentally, one of the world champions in collagen immunochemistry is in Erlangen: Klaus von der Mark. He is still active at the Nikolaus-Fiebiger-Zentrum. I'd say, pick his brains.

Native collagen fibers can be effectively imaged without any staining using second harmonic generation (SHG) microscopy.

The reason for low immunofluorescence signals of collagens is not their poor immunogenicity , no matter how correct it is that collagens are poor immunogens. The reason is that 1) collagen I fibres do not exist. All collagen fibrils or fibres are macromolecular alloys comprising several collagens and also non-collagenous macromolecules. Do not think of a fibre as a heap of collagen molecules (which is what most people do). 2) The latter are enriched in the periphery of the fibres and mask the epitopes of most anti-collagen antibodies. Unmasking is required to obtain good immunofluorescence signals. In some cases, treatment with acid or heat is not sufficient to expose the epitopes but partial degradation by proteolytic enzymes helps in those cases.  3) The exact treatment depends on the tissues and collagens investigated. Literature: Hendrix, M.J.C., E.D. Hay, K. von der Mark, and T.F. Linsenmayer. 1982. Immunohistochemical localization of collagen types I and II in the developing chick cornea and tibia by electron microscopy. Invest. Ophthalmol. Vis. Sci. 22:359-377; Mendler, M., S.G. Eich-Bender, L. Vaughan, K.H. Winterhalter, and P. Bruckner. 1989. Cartilage contains mixed fibrils of collagen types II, IX, and XI. J. Cell Biol. 108:191-197; von der Mark K, von der Mark H, Timpl R, and Trelstad RL: Immunofluorescent localization of collagen types I, II, and III in the embryonic chick eye. Dev Biol 59:75, 1977. Incidentally, one of the world champions in collagen immunochemistry is in Erlangen: Klaus von der Mark. He is still active at the Nikolaus-Fiebiger-Zentrum. I'd say, pick his brains.

Hi Martin,

It would probably be best to explain what you are trying to do and with what tissue you are working.  Also, what have you tried that didn't work so well?  As you mentioned, there are plenty of anti-collagen Ab out there and the fact that collagen is pretty conserved between species really is not an issue with moderns Ab production methods (BTW, collagen in not that non-immunogenic). In immunolabeling, most lack of signal are due to antigen degradation/denaturation.  Are you working with frozen tissues/sections?  Fixation can be the culprit for low signal. Avoid cross linkers if possible or limit the fixation time if you have to use them. Paraffin sections will rarely give you a good IF signal, and give tons of background, because of the harsh fixation.  I am assuming you are doing simple indirect IF.  You can look into amplification steps if you cannot change your specimen processing (https://www.vectorlabs.com/catalog.aspx?catID=40).

There really are many possible reasons you may not be getting a good signal but the lack of affinity of the antibody is not likely to be the problem.  I haven't done it for a while but the Sigma c-2456 antibody has worked well for me in the past.

Good luck!

Thank you all for your ideas.

I am  using rat mesenchymal stem cells which I stimulate for collagen-I production with various media (osteogenic and other) and then image with SHG multiphoton microscopy to study fiber networks with plain cells and cells on biomaterial scaffolds. It would be very helpful to have a verification technique for SHG and that can only be immunofluorescence (IF).

I have tried indirect IF after fixation (10 min formalin), however, I get only very poor signals even though using appropriate primary Abs (Thermo PA5-29569 and abcam ab34710) and very strongly fluorescing Qdot-labeled secondary Abs.

Pepsinization to unmask hidden epitopes within the collagen-I fibers (as suggested by Peter Bruckner) sounds like the explanation for this finding. I will try this.

You'll likely find the answer to  your question on collagen I immunofluorescence in the follwing paper.  2006 Mol. Biol. Cell - Type I collagen is aggregated in endoplasmic reticulum etc - Ishida & Nagata.

All the best

Hi Martin

You can check out this paper. I did stain for collagen-I in bovine aortic tissue. I could not find individual fibers and the reasons for that are mentioned above by Peter Bruckner. If you want to image for the presence of collagen in any tissue the antibody and method is mentioned in the paper. The collagen looks to be in the form of clumps and sheets rather than fibrils.

Hope this helps.

Cheers.

Article Regional variations in the nonlinearity and anisotropy of bo...

Hi!

You wrote that stimulate rats' MSCs, right? Do you differentiate MSCs to specific cell line, e.g. osteoblasts or chondrocytes? If so you should first check specific markers, like for ex alkaline phosphatase, Ca++ conc in case of osteoblastic-like cells.

Before you use IF method to visualize col I it would be worthy first check col I expression level in cells you obtained after stimulation, e.g. by specific sond (Taqman) and standards. The trivial reason you are not able to see col I fibrils might be low expression level...

regards, K.

In the meanwhile I have tried Proteinase K (Dako, ready-to-use, S3020) for partial digestion of fibers to unmask hidden epitopes and in fact signal becomes more intense. MSC`s undergoing osteogenic differentiation appear to produce very very thin fibers while cells in serum-reduced medium with no osteogenic stimulants can be detected and recognized much better. A part of the problem is that fixation of cells partially breaks them open allowing antibody to bind to procollagen monomers in the ER and Golgi which are unmasked and this creates strong IF from within the cells. It is thus hard to detect extracellular fibers in front of relatively high signals from the cells. Confocal microscopy is very helpful to block undesired signal.

Does one of you know if pepsin or trypsin work better to unmask the binding epitopes in the collagen fibers and what conditions (conc., time/temp. of incubation)?

Kind regards,

Martin