Re: immunogold labeling, EM questions

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Elizabeth wrote:
  It's been a while since I have done any EM so I am not really up to date
with techniques, so I have a few questions for anyone interested in

Q1.  In paraffin,  I believe the avidin/streptavidin-biotin method is
considered more sensitive than the direct  method (labeled antibody binds
to antigen). Is everyone using this ABC technique now for immunogold work
as seems like it should be better, but does anyone have first
hand knowledge?
Q2.  In sectioning lowicryl, do you guys have any special methods for
dealing with it. I have never sectioned it.  For .5 um sections do you
still heat the section on a hot plate to spread the section like you would
do with
Q3.  If I am not interested in low temperature, what would be the reason to
choose  LR White over  Lowicryl or vise versa for immuno?
Q4.  More specifically, if anyone out there is using immunogold to identify
collagen, do you recommend using the smaller gold size of 5 or 1 nm vs the
10 nm?
Dear Elizabeth
Hope the following is of help:
A1. Contrary to immunoenzymatic and immunofluorescent immunodetection
systems, the immunogold and gold/silver techniques are based on a
particulate marker molecule. Single gold or gold/silver particles are
visible in the (electron)microscope. By using a biotinylated secondary
antibody and a strept avidin gold conjugate you will not detect more
antigen. You will only see more particles. A possible disadvantage
(certainly on the EM-level) of the three step method is the increase of the
maximum distance between gold particles and antigen. I would therefor
recommend the use of a two-step method instead of the three-step method you
are thinking off.
A2. Depends on what Lowicryl you are using. K4M is more hydrophylic and
atracts water. The water level in the knive boat has to be somewhat lower
than you normally use with hydrophobic plastics (epoxy resins). Cutting
properties of Lowicryl HM20 are comparable to Epon.
A3. Depends on the antigen (and primary antibody). Sometimes LR-white works
better, sometimes Lowicryl. Polymerization of these type of plastics is
exothermic, so polymerization at 50oC does not mean that the  specimen temp
is 50oC. This might have a negative influence on antigenicity. Low temp
polymerization might be the solution in that case.
A4. In general, labeling density increases with decreasing gold particle
diameter. It depends on the amount of antigen available which size is most
Regards, Peter

Peter van de Plas
Costerweg 5
6702 AA Wageningen
The Netherlands
phone: (31)-317-497676
fax: (31)-317-415955

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