Is there a problem because the antibody is raised in mouse?
We used polyclonal antivGluT2 from guinea pig (diluted 1:500; AB5907, Chemicon). Yes, mouse antibodies used in mouse tissue can result in a high background. You have to increase the number of washing steps, include some Triton in the buffer. If that does not help, there are "mouse on mouse" kits commercially available that prevent your secondary anti-mouse antibody to bind endogenous mouse Igs.
There is also a good VGluT2 monoclonal available from NeuroMab that works well for immunostaining. We have not experienced exceptionally high non-specific background in the brain tissue itself. Because the brain is immune-privileged by the blood-brain barrier (also called the neurovascular unit), there is little endogenous immunoglobulin in the tissue to produce high levels of non-specific background labeling, that would characterize other tissues and organs. However, we do observe non-specific labeling of blood vessels due to the goat anti-mouse secondary antibody recognizing endogenous mouse immunoglobulins in the serum. We do not perfuse our tissue, so the serum and blood remain in the vessels. Perfusion fixation would be expected to eliminate the non-specific vascular staining.
There are also VGluT2 antibodies raised in non-mouse species that work well. Cemicon (Now sold by Millipore) had a pretty decent rabbit anti-VGlut2. Synaptic Systems offers VGluT2 antibodies raised in 3 or 4 different hosts.
Hi Johanna. If you are looking for suitable antibodies i recommend checking www.pAbmAbs.com. Here, scientists can share information about antibodies they use in their research and i can see several different antibodies Vglut have been reviewed, includingthis:
http://pabmabs.com/wordpress/?p=508
Even though this is against rat it apparently also detects mouse and by using this you will avoid problems regarding mouse on mouse.
I hope this helps. I also hope you will review the antibodies you use in your research :)
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