Antibody for chicken endothelial markers

[albystone]

I'm working with the chicken embryos.
Does anyone know a good commercial antibody specific for the chicken endothelium (like CD31, FVIII, Endoglin, or any other possible marker) and suitable in IHC (formalin fixed, paraffin embedded tissue sections)?
Please help me!  
Thanks.

Alberto.

[davidtest]

Easy question but hard to find answer.

I have done extensive search from 5 antibody search engines (http://www.ihcworld.com/antibody.htm#AF) and did not find any specific anti-chicken endothelial cell markers.

However, here is a link to the list of all endothelial cell markers available from Chemicon: http://www.chemicon.com/techsupp/endothelial.asp
Some of them are specific for embryonic endothelial cell such as TIE-1 You may find a closest one to try. Good luck.

David

[albystone]

Sorry if I reply only now.
You're right when you say that it's hard to find answer.
Thank you very much for the help.

Alberto

[Belkosky]

DakoCytomation's polyclonal anti-Von Willebrand Factor (Factor VIII, A0082) has a strong documented cross-reactivity with chicken.

Dan

[albystone]

Thank you for your reply.
I tried to use the antibody some time ago.
I used the microwave method (Citrate Buffer) to unmask the antigen (as suggested in the antibody datasheet) without results.
After your reply I found a different method for the antigen retrieval in another datasheet (code n° T1100, always DAKOCytomation).
So I use the suggested enzyme predigestion (with trypsin) with discrete results.

Any suggestion about  the optimization of the staining?
Thanks.

Alberto.

[ihcwor2]

In my experience, endothelial cell markers such as CD31, CD45, Factor VIII, etc. work better with Zinc fixative along with trypsin digestion. I have been tried using pronase digestion and it also worked well.

Here is a protocol for Zinc Fixative:

1. Tris buffer (0.1M, pH 7.4):
     Tris Base ------------------- 12.1 g (TRIZMA)
     1N HCL ----------------------81.5 ml
     Distilled water ------------- 900 ml
     
2. Zinc Fixative:
      Calcium Acetate ---------------------- 0.5 g
      Zinc Acetate -------------------------- 5.0 g
      Zinc Chloride ------------------------- 5.0 g
      0.1M Tris buffer made above ------- 1000 ml
Mix to dissolve. The final pH will be approximately 6.5-7.0. Do not readjust the pH, as this will cause the zinc to come out of solution.

3. Store Zinc Fixative at room temperature.

4. Fix tissues for 24 hours and proceed to routine paraffin embedding.

Reference:
Nitta,H. etal. 1997. Imporved InSitu Immunodetection of Leukocytes on Paraffin-Embedded Mouse Spleen. Cell Vision 4: 73-80

You can also purchase Zinc fixative from BD Biosciences if you like.
IHC Zinc-Fixative, BD Biosciences, Cat# 550523

And finally play around with different dilutions and I prefer to use high concentration for this kind of markers.

Hope this helps.

Richard