For the last few weeks, I've been attempting to stain my brain sections for CD3 and CD68 to identify/quantify the cells that comprise of the infiltrate in my GM-CSF -/- mice. It had been working well, but we found that using the rabbit anti-human CD3, although the antibody was specific for the right cells, there was background staining of residual plasma in the blood vessels as well as staining cells that shouldnt have stained within the cerebellar white matter.
Today and yesterday, I attempted to get rid of both of these. Yesterday I changed the secondary antibody from the DAKO LSAB kit to the old skool swine anti-rabbit and streptavidin, as well as doing an anti-isotype control. The anti-isotype *worked* in the sense that nothing came up, but since the normal run showed absolutely nothing at all when it should have, I consigned both runs to the bin. Today, after talking to Tech Support from DAKO, I added mouse serum 10 microlitres/ml of LSAB Universal Link solution as well as the anti-isotype control. Both worked (yay!) in that the plasma staining is gone, but the other cells remain.
How puzzling. Maybe there are endogenous CD3 elements in the CNS. That's intriguing.
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