Dearest hive mind,
I have been trying to get a staining going for collagen type II in human specimens (de-calcified). Until now, without great results. My initial approach has been with enzymatic antigen retrieval with pronase and hyaluronidase according to Amirtham et al. 2019 (1 mg/mL Pronase for 30 min. and 5 mg/mL Hyaluronidase also for 30 mins). I had a suspicion that the enzymatic treatment would be quite rough on my sections (formalin fixed, paraffin embedded), so I did not do 37 degree during incubation with the enzyme. Of course the sections basically fell of (I guess the enzymatic treatment was too harsh). But, the most annoying thing was, that the tissue that was still on the slide, did not stain nicely (bone with clear areas of cartilage (Specimens were demineralized in 10% EDTA ). I used Dako Envision and AEC as chromogen (In my old lab we used DAB so this is a first time for me). The antibody is a polyclonal rabbit anti human Collagen II from Abcam. The incubation time at room temp with the primary antibody was 2 hours. Do any of you genious-researchers have any good ideas about what I am doing wrong. I'm used to looking at histochemistry, and boy, with a bit of Picrosirus Red you are not in doubt about what is collagen and what is not - but maybe I should not expect to see an intense staining. What I am ultimately trying to do, is to show where there is Collagen I and where there is collagen II as a way of saying "this is cartilage as opposed bone" - am I approaching this from a wrong angle?
Dear Sune,
Col II in my case worked very well.
Col II Antibodies source: https://dshb.biology.uiowa.edu/search?
Decalcification 10%EDTA or mild formic acid both worked. Here is my full IHC protocol (enclosed) and antigen retrieval mentioned in the paper (enclosed)
If you have any further question, pl let me know,
Subhash
Dear Tune,
I assume that decalcification disturbs your immunostaining for collagen II. Try normal cartilage (with out previous decalcification) to find out.
Best wishes
Daniela
Dear Sune,
decalcification should not disturb COLII-IHC as far as our experience goes.
I also work with human cartilage specimen and know what it fells like when the sections just float off the slides during IHC.
The most important thing is that you use coated slides! Either silanized slides (it is cheap, but more work for you) or you use slides from commercial sources (i.e. "super frost" slides; these are more expensive). For antigen retrieval, we perform a digest with pepsin (1 mg/mL in 0.5 M acetic acid) for 30 min at 37°C and a primary antibody from Acris (AF5710; incubation over night at 6 °C). Afterwards we stain with the Dako LSAB2 System-HRP kit (advantage: biotin & avidin-based -> signal amplification).
If you just want to differentiate between cartilage and bone, you might also perform a Safranin-O/ fast green staining: this stains the proteoglycans of the cartilage nicely red, while the bony parts appear in green/blue. It is much easier!
Attached you will find a publication in wich we used both staining methods in an in vivo cartilage-trauma model after decalcification (rabbit model; but we used the same COLII-antibody as for human specimen).
Best wishes
Jana
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