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

Mon Mar 21 15:03:29 CST 2005

Thanks Gayle for the protocol and
suggestions...looking it (and other protocols) over I
had two more questions:

1) why are gold conjugated Abs light sensitive?  Your
protocol has the incubation in secondary in the
dark...I can understand why some silver developers are
light sensitive...but what's the reasoning behind
keeping the secondary incubation in the dark?

2)  are gold conjugated Abs more sensitive to
detergents?  Your protocol has both Ab incubations in
PBS (no detergent)...and another protocol for
immunogold had the primary in PBS with detergent..and
then specified (!!!) no detergent in incubation with
the gold-labeled secondary...For all my other ICC
(usually avidin-biotin-peroxidase detection) I have
detergent in every incubation step...

I'm planning on doing another run to test the effects
of light exposure versus foil wrapping and detergent
versus no detergent, so maybe that will shed some more
light on the source of my immunogold problems...I'm
also going to take up your suggestion of contacting
Chris van der loos, I just wanted to broaden my base
for answers by including my questions to the Histonet
subscribers...

and if anyone has encountered any problems with
immunogold labeling for light microscopy please let me
know what they were and how you solved them...

Thanks again for all the help...
Adam
Department of Physiology and Biophysics
University of Illinois
Chicago, IL

--- Gayle Callis <gcallis <@t> montana.edu> wrote:

> Enhanced cold particles were aways very black and I
> used a nuclear fast red 
> counterstain.   I do not recall ever getting the
> colors you describe.  I 
> purchased some of my BBI  gold conjugates (1 nm size
> particles) from Vector 
> but they do not seem to have these anymore.  I do
> not do the kind of 
> immunostaining you do ( transcription factor in
> brain) so I couldn't 
> comment on any of that in terms of immunogold.
> 
> If you have an epiillumination microscope, take a
> look at your sections.  I 
> strongly suggest you visit with Chris van der Loos
> on this, I think he can 
> help you with this more than I can - it really has
> been a long time ago 
> since I did it.   I have attached my IGGS protocol,
> but if you need silver 
> enhancing reagents, I would have to retype these.  I
> do not have these on 
> file as of now.  Mine was a silver acetate/ citrate
> buffer, and very dicey 
> to work with, if the enhancement needed longer time,
> I would have to go to 
> another fresh enhancing solution, as it broke down
> rapidly, and often three 
> changes.   These reagents were very fresh before
> use.  I used o close and 
> lock my door plus never answer a phone when I did
> this step.
> 
> Attached is an old IGGS protocol I used, just for
> comparison sake.  I 
> remember having stringent requirements for the
> buffers I used.
> 
> A tough call, but talk to Chris - he had great
> success with his IGGS, and 
> he combined it with a double stain, for very nice
> colocalization purposes, 
> spectacular.
> 
> 
> 
> 
> At 02:44 PM 3/18/2005, you wrote:
> >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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> >
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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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