[Histonet] IHF with blue conjugated secondaries

From:"Danielle Crippen"

Dear All,

I have an investigator who is in need of triple fluorescence labeling on mouse brain tissue.  The green fluorophore is injected and the request is that we double label for two additional antigens.  

Our problem is that we cannot find a blue conjugated secondary that works at all.  We've purchased AMCA Dn x Rb (Jackson) and 350 Dn x Gt (Molecular Probes) and have tried them at every possible working concentration recommended on the spec sheets.  We've included GFAP positive controls with Cy3 secondary (this works beautifully) and we've tried using alternate tissue to eliminate the possibility that it's our particular sample.  We've also tried varying the blocking method and several different protocols.  

Considering the confines of our microscope set-up, we must use a blue and a red conjugated secondary.  Additionally, one of our primary antibodies is made in goat, so both our secondaries must be made in donkey.

Any suggestions as to vendors, protocols or particular references from the literature are VERY welcome!!!

Many many thanks in advance!!

Danielle Crippen
Morphology Core Manager
Buck Institute for Age Research
8001 Redwood Blvd.
Novato, CA 94945

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