Dear Andrew, In my experience most of the highly cross-absorbed antibodies will still give you some background staining, either reacting with unstained cells or partially recognising primary antibodies of species they should not recognise. The level of background I am taking about is usually around 1%, therefore depends of your application if you can live with it or not. Higher background can be reduced with added washes, but more you wash and more cells you loose. If you are planning multicolour experiments with primary of different species followed by specie-specific secondary, do the experiment: label unstained cells with your secondary and check. Also label another lot of cells with the primary of specie A + secondary anti-B. If your primary are of the same specie and same isotype you need to use the Zenon system (Molecular Probes) or Fab fragments (Jackson immunochemicals), and both website have specific staining protocol that usually includes one or more blocking steps. If you are planning a two-steps followed by directly conjugated primaries the answer is yes, you will have to block, and with IgG of the same species the first primary was made in, to block all the free unbound epitopes of your secondary. Hope this help Best regards Mara Mara Rocchi BVM&S, PhD Flow Cytometry Manager Moredun Research Institute Pentlands Science Park Bush Loan, Penicuik EH260PZ Scotland, UK Think before you print. Save paper and help the environment-----Original Message----- From: Andy Hoffman [mailto:andrew.hoffman@tufts.edu] Sent: 07 January 2008 20:08 To: cyto-inbox Subject: alexa fluor A simplistic question from a relative novice flower: Do polyclonal secondaries conjugated to Alexa Fluors (e.g. Alexa Fluor 647 goat anti-mouse IgG (H+L) A21236 - InVitrogen/Mol Probes) that are supposed to be 'highly absorbed' against alternative species (e.g. rat) actually cross-react with rat anti-mouse secondaries at certain concentrations? Can they pick up unstained cells in the absence of other primaries/controls? Lastly, is it suggested to wash after the first round of primary + secondary-alexa, then block with normal mouse Ig as previously mentioned in the CD133 string, before adding other primaries? Thanks so much for your time Andy -- Andrew M. Hoffman, D.V.M., D.V.Sc., Diplomate, A.C.V.I.M. (Large Animal Medicine) Associate Professor Cummings School of Veterinary Medicine Tufts University Director, Lung Function Testing Laboratory Department of Clinical Sciences Bldg 21, Suite 110 200 Westboro Road North Grafton, MA 01536 andrew.hoffman@tufts.edu Phone (508) 887 4589 Fax (508) 887 4590Received on Wed Jan 9 14:18:00 2008
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