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(Q) Problems w/silver enhancement of gold particles

Kenneth J McNeil mcnei002 at maroon.tc.umn.edu
Fri Mar 8 16:05:31 EST 1996


I have been attempting to localize floral-specific gene products in
tomato flowers using silver enhancement of gold particles. The problem is
I have been getting a relatively high background (browning of tissue and
black particle formation all over the slide).

I had heard of photosynthetic tissue reacting with the silver-enhancing
solution, but haven't been able to find any references listing possible
solutions to the problem.

Conditions:

10 micron cryosections of tomato flowers fixed in HistoChoice (Amresco)
and mounted in OCT. Sections placed on gelatin subbed slides. Tissue
blocked in 5% nonfat dry milk in Tris buffered saline, pH 7.5, with 0.1%
Tween-20 (TBST) for 1 hour. Rinsed 3x5' TBST. Primary Ab, 1:200 dilution
(Ab raised in rabbits against bacterial fusion protein [pET28a fused to a
floral specific gene]), 1 hour in TBST. Rinse 3x5' TBST. Secondary Ab,
1:80 dilution, goat antirabbit with 4 nM gold conjugate in TBST, 1 hour.
Rinse 3x5' TBST. Rinse PBS 2x5'. Fix with 2% glutaraldehyde in PBS, 15'.
Rinse 2x5' PBS. Rinse 2x5' di H2O.
Silver enhancement solution (silver acetate solution mixed with
hydroquinone in citrate buffer at a pH of 3.8). Detection time is
approximately 18 minutes at 23C. Slides kept in darkness during silver
enhancement. Slides rinsed di H20, placed in fixative 2', rinsed di H20
5'. Viewed under light microscope.

Thank you for your help,

Ken McNeil
Horticultural Science Department
University of Minnesota                         mncei002 at maroon.tc.umn.edu



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