[Histonet] Using Tween 20 with my IGSS method

[Gayle Callis]

Adam,

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 
protocol.

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 
microscopy.


>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
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)