alexa fluor

Mara Rocchi Mara.Rocchi at
Wed Jan 9 02:43:26 EST 2008

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 Rocchi BVM&S, PhD
Flow Cytometry Manager
Moredun Research Institute
Pentlands Science Park
Bush Loan, Penicuik
Scotland, UK
Think before you print. Save paper and help the environment-----Original
From: Andy Hoffman [mailto:andrew.hoffman at] 
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


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 at
Phone (508) 887 4589
Fax (508) 887 4590

More information about the Cytometry mailing list