[Histonet] Using Tween 20 with my IGSS method
I went back and reviewed my IGSS methods, and why I had used Tween in some
steps and then not with secondary and rest of protocol.
At 02:03 PM 3/21/2005, you wrote:
>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?
**** I think that is a typo error when I typed up that protocol flow
sheet. Protection from light was when silver enhancer was made up and
subsequently used. Somehow, I don't recall ever protecting gold conjugated
secondary antibodies from the light but if I did, it would not have hurt
what I did.
>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 was working with the Microprobe system (mentioned in Step 1 of protocol)
to do primary incubations, and all steps before and with primary antibody
application had to contain Tween 20 in order to make the capillary gap
work. After rinsing (via wicking, etc) primary away with buffer containing
Tween 20, I got rid of the detergent to finish out the protocol without it.
The secondary diluent did not contain Tween nor did the rest of the
It is important to reduce ionic interactions protein protein with IGSS to
prevent background but my use of detergents was not for that purpose. If I
had to do all this over again, I think I would go purist, and do IGSS
without detergent involved, just stringent buffer/diluents/gelatin/BSA
additives seen with IGSS staining methods. My hard copy file folder
floweth over with information!
I have this article by van der Loos CM and Becker, J Histochem Cytochem
42(3):289-295, 1994 (a superb article about epi illumination with a double
stain using IGSS and IHC alk phos methods). Stirling did a large study on
detergents with IGSS, published in J Histotechnology. If you have a chance
and can find it, there is a whole special issue on doing immunogold
staining in J Histotechnology.
Not sure if Tween really makes a difference in success of IGSS, maybe it
does. I would like to try Aurion enhancing kit for 5 to 15 minutes versus
the long, painfully (closed doors, never answer phone!) tedious silver
enhancement method I did using inhouse reagents. I keep encouraging my
immunologist to try IGSS, but he doesn't have epi illumination which is
truly the ideal way to view these stained sections over standard light
>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
>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...
>Department of Physiology and Biophysics
>University of Illinois
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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