Hallo Shi, if your positiv control is treated in the same way as your brain tissue and you have a positiv result in your control material and nothing in your brain tissue, you have to check weather the control tissue is also from rats. If not the rusult shows you that this CD31 antibody is not suitable for rat tissue.  If your control tissue is from  the same species like the brain tissue, in that case the result shows you that the protein CD31 is not detactable in brain tissue. That is exactly the reason why you are running positiv controls. Positiv controls show you that the immuno protocoll is woking even when you could recieve negativ results. In that case  you have to look for another CD31-AB wich is suitable for your kind of tissue.

Hi Ute,

Thank you for your reply. Both my positive control and control tissue are from rat tissue. I read online that certain methods of extracting and fixing the brain can affect staining of blood vessels, and was wondering if anyone knew the best way to prep brain samples before staining.

Hi Beverley,

I am using frozen sections, and I didn't do antigen retrieval because I thought that was only for paraffin embedded sections. Or is that not the case?

Hi Shi,

you can do antigene retrieval on frozen sections. The sections have to be mounted on Superfrost Plus slides.  You can treat them in the same way you do it with paraffin sections. In my case I prefere the IHC with free floating sections. Some enhancement methodes: 1. Streptavidin labeled with fluorophores or with POX is more sensitive the labeled secondary antibodies.  But for  Streptavidine you need biotinylated secondaries. 2. Strepavidine -Flu - Anti-Streptavidine - Strepavidine -Flu. 3. Biotinylated Tyramine enhancement (Adams JC et al. J Histochem Cytochem 1992;40:1457-63). All this techniques are working on free floating sections as well as on paraffin or mounted frozen sections.