[Histonet] Re: CY2/CY3 vs Alexa488/546-555

Gayle Callis gcallis <@t> montana.edu
Thu Dec 22 10:11:09 CST 2005

To get around the problem of Alexa conjugated secondaries you don't like 
from Molecular Probes,  buy your favorite secondary from Jackson, and 
conjugate it yourself with an Alexa dye conjugation kit from Molecular 
Probes.  You will get the best of both (of your favorite) worlds.  The kit 
conjugates three antibodies, and is a very simple to use.  This may be the 
only way to guarantee the quality you desire and still have your favorite 
Alexas available.

We have experienced similar problems as you with MP Alexa conjugated 
secondaries.  However, last week's IFA using a highly adsorbed goat 
antiRabbit-Alexa 488 at 0.5ug/ml with a rabbit polyclonal for a bacteria in 
frozen mouse lung sections gave spectacular results - a pleasant surprise 
for me and one PI.

At 05:40 PM 12/21/2005, you wrote:
>Has anyone done a SIDE-BY-SIDE comparison of Cy2 or Cy3 as compared with 
>Alexa 488 or Alexa 546/555? I am ideally looking for experimental results 
>not anecdotal information if possible .... I know we have all heard that 
>Alexas are the best and this is certainly true as compared to FITC. But 
>are they better compared to CY2 and CY3 from Jackson Labs?
>I ask b/c I love Jackson Labs secondaries and they don't sell Alexa 
>conjugates :( I use Alexa Streptavidin from Molecular Probes but am not 
>such a fan of their secondaries for TISSUE staining (they give me some 
>strange artifacts sometimes although they work great for cell culture 
>work). I want to keep ordering Jackson ImmunoResearch quality 
>secondaries  - they are just SO SPECIFIC for mouse work but they only sell 
>CY2 and CY3 (which have worked for me thus far).
>Decisions, decisions .... Comments, opinions, suggestions?

Gayle Callis HTL, HT, MT(ASCP)
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University
Bozeman MT 59717

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