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

[Adam Perry]

I actually started with in house prepared
reagents...tried every protocol I could get my hands
on and none of them really worked well and all were
time consuming to prepare...so I broke down and bought
a kit (from Aurion) and it was much easier to use, but
didn't seem to work.  I haven't tried the
epi-illumination scope yet though.  

What did your tissue sections look like after IGGS? 
In the original lab I trained in, the sections would
go through a color change from yellow, to brown, to
greenish brown and then we'd stop the silver
enhancement.  The antigen I'm staining for is CREB
(transcription factor found all throughout the brain),
so that may be why the entire section went through the
color change...but the staining I did with the Aurion
kit...the tissue looks amazingly clean, no background
staining...or anything...is that typical with IGGS?

Thanks again,
Adam 
Department of Physiology and Biophysics
University of Illinois
Chicago, IL 
--- Gayle Callis <gcallis <@t> montana.edu> wrote:
> 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)
> 
> 
> 


		
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