Re: triple labeling with antibodies

Posted by Jeremy Keith Brown on
URL: http://confocal-microscopy-list.275.s1.nabble.com/triple-labeling-with-antibodies-tp591231p591237.html

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If you do go down the route anti-mouse/anti-rat route you will face  
cross-reactivity issues. As Martin says, mouse cross-absorbed anti-rat  
polyclonal (and vice versa) secondaries aren't great, even from  
Jackson. However, there are some very good mouse anti-rat monoclonals  
available (try Serotec, BD or SouthernBiotech) which if used in the  
right order will work for you. Basically you need to label you samples  
sequentially: mouse and rabbit primaries; then goat anti-mouse and  
anti-rabbit secondaries; then incubate the samples with 10% normal  
mouse serum to block any free goat anti-mouse paratopes; then your rat  
primary (in 10% mouse serum) followed by the mouse anti-rat secondary  
(in 10% mouse serum). Alternatively, you could indirectly label the  
sample with the rat and rabbit primaries, block with mouse serum and  
then come in with the mouse primary labeled with your choice of Alexa  
flour conjugated Zenon FAB fragments. Obviously, you will need single  
positive controls for each primary to confirm you're not just  
detecting spurious cross-reactivity.

Cheers,
Jeremy

Dr Jeremy K Brown
The University of Edinburgh
Easter Bush Veterinary Centre
Easter Bush
Midlothian
EH25 9RG

Tel:  ++44 131 650 7348




On 14 Dec 2007, at 15:18, Martin Wessendorf wrote:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Shalin Mehta wrote:
>
>> We were considering purchase goat polyclonal for our antigen, but  
>> it seems we will be better off with rat polyclonal and using highly-
>> adsorbed goat secondaries against rabbit,mouse and rat. Zenon kit  
>> and Fab fragments also look useful, but will need to read up on them.
>
> There are many different ways of getting satisfactory labeling.  My  
> own recipe for triple-labeling uses Cy2, Red-X (or Cy3, depending on  
> the experiment) and Cy5 secondaries (--I don't use Fab fragments)  
> from Jackson ImmunoResearch that have been cleaned up for minimal  
> cross-reactivity.  (--though you do NOT want to use anti-mouse with  
> minimal cross-reactivity against rat unless you have to use rat as  
> the source for one of your other primaries.  Rat and mouse are  
> immunologically so close that cleaning up against one takes a big  
> bite out of the labeling intensity for the other.)  I'd use the Cy2-
> labeled secondary in combination with your strongest primary, since  
> the Cy2 signal is a bit weaker than the others.  I would then run  
> the tissue through alcohols and mount in DPX, which gives you a  
> permanent, dry mount.  (I like DPX for those features.)  I've heard  
> others say that they get rapid photobleaching with DPX but I have  
> tissue mounted in it that's been sitting by my microscope for almost  
> 15 years and that I use periodically as test-slides.  If you don't  
> blast it with high illumination power at 60x, the labeling lasts  
> just fine.
>
> Why do you need spectral imaging if you're just looking at dead  
> tissue or cells and the dyes are as well separated as you're  
> describing?  Or is this for when you throw GFP  into the mix?
>
> Good luck!
>
> Martin
> --
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