You could use a mixed goat and rat serum blocking to reduce cross species recognition, but considering how easy it is to label your antibody directly with either PE or APC using for example the Innova biosciences kits I would go that route instead, unless you absolutely need those colour channels for other antibodies. There are still unwanted interactions via Fc receptors, anti species reactions, rheumatoid factors, fibrinogen etc and entrapment in dead cells to be considered. Gerhard Nebe-von-Caron Research Scientist and Biomedical Engineer SPD-Spark Swiss Precision Diagnostics Priory Business Park Bedford, MK44 3UP, UK Tel +44(0)1234-835474 Fax +44(0)1234-835002 mailto:g.nebe-von-caron@spdspark.com -----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 Fri Jan 11 13:18:00 2008
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