I have a question about the A2B5 antibody and its use for staining oligodendrocyte precursor cells, that I'd be really interested to hear your experiences and expertise about. I’m aware that A2B5 should be used on live, unfixed, non-permeabilized cells because it reacts within intracellular antigens. When I’ve stained OPCs with it I only see faint punctate staining at the cell surface. In lots of papers however, A2B5 staining is bright and continuous throughout the cell. Is this because of the staining method (I note a lot of people seem to use fixed cells, which could also of course be permeabilized so the staining could possibly be artifactual). Or could it be that in the cells at that time I look at them A2B5 is being down-regulated? I have not co-stained the cells but I do also see O4+ cells in the prep at the same time. Also, has anybody noticed any differences between A2B5 supplied from different suppliers? I have so far used it from Abcam, but I note you can get it from Sigma/R&D etc.
Hi Mark, I have used the A2B5 from Sigma and it stains very well. I would suggest to do blocking and primary in DMEM/10%NDS (normal donkey serum). Fix with 4%PFA after you have stained with secondary. We recently published a paper in PNAS showing A2B5 staining in the optic nerve at 7DIV (see Nikolakopoulou et al., 2013). Please let me know if you have any more questions
Best,
Mariangela
Mark, This is a fixation and staining protocol we've used very successfully with A2B5 from Chemicon (now Millipore), a mouse monoclonal (original publication was Mi and Barres, J Neurosci 1999, 19(3).
On progenitor cells in vitro, fix cells for 30min in 4% PFA at room temp, wash 3x10min with PBS, block in either fish skin gelatin buffer (1%BSA, 0.2% fish skin gelatin, in TBS) or donkey block (3% donkey serum in TBS). NOTE: no TX-100!!
Use primary AB 1:100-250 in blokcing solution, incubate at 4° over night. Worked great! Sample image attached (from Engelhardt et al., 2004, Exp Eye Res 78). Good luck!
Hi guys, sorry for my late reply! Thanks very much for your answers, I really appreciate your expertise. Great work in your papers too! Maybe catch up with you again soon. Best wishes, Marc
Hi guys, I tried the Sigma A2B5 today and it worked great on live cells! Previously I'd used Abcam's but it gave faint-to-non existent staining! Thanks for the advice!
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