[Histonet] Problems with Gold-immunolabeling and silver enhancement in brain tissue

[Gayle Callis]

How are you doing the silver enhancement, from inhouse prepared 
reagents?  We found making up enhancement reagents inhouse a lot of work, 
then it was hard to control the enhancement, a painstaking procedure.

Dr. Chris van der Loos uses a silver enhancement kit from 
http://www.aurion.nl .  When I visited his lab, this was a snap to use the 
Aurion kit and we had excellent results.  For visualizing IGGS staining, 
epi illumination is superior than trying to find the black enhanced 
particles via standard transmitted light microscopy.  You may have 
staining, but can't see it very well -  however with epi-illumination, the 
enhanced gold particles show up like little light bulbs!!

You can also ask him about his immunogold methods at 
<c.m.vanderloos <@t> amc.uva.nl>.

At 12:16 PM 3/18/2005, you wrote:
>I'm trying to perform silver enhancement of my gold
>labeled secondary antibody (1.4nm gold) in an
>immunocytochemistry (ICC) protocol in rodent brain
>tissue.  I know that the primary antibody and initial
>steps are working because when I use an
>avidin-biotinylated peroxidase kit to visualize a
>biotinylated secondary antibody (as opposed to using
>the gold conjugated secondary antibody)I get great
>labeling of my antigen.  I've done a dot blot with the
>gold labeled antibody and I seem to get silver
>enhancement of particles only in the dot blot with the
>gold-labeled antibody (as opposed to unlabeled
>antibodies)- so I'm pretty sure there are gold
>particles attached to the IgG.
>
>Are there any obvious differences that need to be
>considered when working with a gold-conjugated
>antibody as opposed to antibodies that are
>biotinylated or conjugated to enzymes??
>
>Any insights would be greatly appreciated..
>Thanks,
>Adam Perry
>Department of Physiology and Biophysics
>University of Illinois
>Chicago, IL
>
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Gayle Callis
MT,HT,HTL(ASCP)
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367 (lab with voice mail)
406 994-4303 (FAX)