3/5/97
Greetings,
I have a user of our EM Unit who wishes to use Fluoronanogold (Nanoprobes)
with Silver enhancement for pre-embedding immuno EM. We will be using
frozen 8 micron sections which will then be PLP fixed (parform + Lysine +
periodate) prior to labelling.
After labelling, we want to post-fix them in OsO4 (as membranes are
important), dehydrate and embed in normal epoxy resin. I have heard that
the Flouronanogold is not stable at 60oC, the normal curing temperature of
the resin.
Has anybody had experience with pre-embedding with Fluoronanogold?
Will Fluoronanogold survive the epoxy resin curing process at 60oC?
If it wont survive, which resin and curing procedure would be the choice to
use in this case? (Quetol 651, Lowicryl?)
Many thanks,
Allan Mitchell.
I already replied privately before I got down our in-box to the MSA
posting. For anyone interested in this, here are our ruminations...
Generally, we recommend low-temperature polymerization resins such as
Lowicryl, LR White or any similar medium for embedding Nanogold or
FluoroNanogold-labeled specimens. This is because some early experiments
with Nanogold-labeled specimens suggested that the higher-temperature
embedding (60°C) might cause the gold particles to be displaced from their
binding sites. However, subsequent experiments with Nanogold in solution
showed that it could be heated even up to 100°C for 1 h with minimal
decrease in optical density; generally, avoiding low pH values (below 7) or
high ionic strengths (0.2 M NaCl or higher) helps ensure its stability.
Because it is smaller than most colloidal gold and has no tendency to stick
electrostatically to proteins or cell components, it may be more free to
move: fixing with glutaraldehyde helps counteract this, and with Nanogold
we also recommend silver enhancement before embedding if it is practical.
There is a section on this in Hainfeld and Furuya's chapter on the silver
enhancement of Nanogold and undecagold in M. A. Hayat's recent book,
"Immunogold-Silver Staining: Principles, Methods and Applications" (CRC
Press, Boca Raton, 1995; pp. 71-96. Check pages 92-92 for the effects of
heating Nanogold. From this section, heating at 60°C for 250 minutes
resulted in a reduction of the optical density to 80% of its initial value
- suggesting that Nanogold can survive most 60°C embedding procedures.
The gold particle in FluoroNanogold is Nanogold, and exactly the same
applies to this probe. However, silver enhancement quickly removes the
fluorescence, so only do the pre-embedding silver enhancement if you have
completed the fluorescence microscopy.
We keep a list of answers and suggestions to frequently-asked questions in
the Technical Help section of our web site:
http://www.nanoprobes.com/Tech.html
All the suggestions made there about Nanogold use and stability also apply
to Fluoronanogold, since these probes use the same gold particle.
Hope this is helpful,
Rick Powell
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If you are speaking of whether the Fluorescent signal of
Fluoro-Nanogoold survives the answer is no, but not because of the 60 degree
heat. The fluorescence is quenched or covered up by the silver enhancement
process. If you want to see the label prior to silver enhancement that is
no problem. I often do this prior to running the silver enhancement to see
that the reagent has gotten in to the sample and gives the expected signal.
Your other choice of resins are lowicryl which you can polymerize at -35
degrees under UV. The FLUOR tag survives this just fine. In regard to
osmication, this has to be done with 0.1% osmium at 4 degrees or on ice for
about 30 - 45 minutes. Standard osmication procedures will reduce the
silver shell that forms around the nanogold particle. This appears as a much
less electron dense cloud or ghost. I have used these reagents with spurr,
epox 812, lowicryl and LR White without any problem.
Joe Goodhouse
Confocal / E.M. Lab
Molecular Biology
Princeton University
jgoodhouse@molecular.princeton.edu