RE: [Histonet] IHF with blue conjugated secondaries

From:"Min-Min Lu"

Hi Danielle,

I have worked out triple fluorescence labeling on mouse tissue with Blue 
fluorescence(AMCA Streptavidin SA-5008 Vector) Red and Green fluorescence 
(Molecular Probes). One of three primary antibodies is made in goat. I 
stained this Ab first. I used Biotin. Anti-Goat IgG (from horse BA-9500 
Vector) as second, then Streptavidin AMCA. After I seen blue positive 
staining I blocked slides with goat serum then use other two primary Ab 
(from mouse and rabbit). The send Abs are from goat. I have tried Donkey and 
chick second. The result is not stable.

MinMin Lu
Molecular Cardiology Research Center
University of Pennsylvania
421 Curie BLVD
Philadelphia, PA 19104

>From: "Danielle Crippen" <>
>To: <>
>Subject: [Histonet] IHF with blue conjugated secondaries
>Date: Wed, 14 Jul 2004 15:39:30 -0700
>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 
>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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