Dear all,
I have a slightly odd question regarding immunogold silver enhancement. I am aiming to look at 2 targets in the same tissue section, but I need a way to differentiate them using metal tagged antibodies. I have unlabelled primaries of different host species and 2 secondary antibodies that are tagged with gold nanoparticles.
Is it possible to silver enhance one secondary antibody, wash away the enhancement reagents and then proceed with the next secondary incubation? In this way I would hope to have one target labelled with gold/silver and another target labelled with only gold.
I am only a Masters student so budget is limited - purchasing any more reagents for alternative approaches such as MaxPar kits or silver tagged antibodies isn't really an option.
Please let me know!
Kind regards,
Charlie
Yes, it is possible. Yi et al. described a double labeling procedure using silver enhancement: " Differentiation of the double labeling was achieved by incubating with one ultrasmallgold conjugate, followed by silver enhancement, and then incubating with the second ultrasmall gold conjugate, followed by additional silver enhancement. This resulted in twogroups of silver-enhanced particles: smaller particles enhanced once and larger particlesenhanced twice." (Yi et al. 2001, "A Novel Procedure for Pre-embedding DoubleImmunogold–Silver Labeling at the Ultrastructural Level" ). In your case you can label your first target (primary + secondary), apply a silver enhancement, and label with the second target (primary + secondary) without silver enhancement. You can also use two secondary antibodys with different sized gold-nanoparticles to differentiate your target structures.
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