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Thanks to all for thoughts thus far.  I should clarify my question a little.
I am confident that our protocol is labeling the antigen (VE-Cadherin aka
cadherin-5) well enough, i.e., neither penetration nor epitope access is a
problem.  What I want to improve is spatial resolution.  The images are
"fuzzy" for want of a better word.  I believe that it is a problem of light
scattering.  I did a few preliminary experiments for another researcher
about 3 years ago in which we labeled endothelial cells in perfused muscle
with a plant lectin and also used rhodamine phalloidin to bring up the
muscle fibers (modeled on one of your papers, Fay).  For that protocol we
were able to use methyl salicylate to clear the tissues and it really
improved the quality of those images.  For my current application I just do
not know if there are any "clearing" protocols known to work without
diminishing antibody binding.

  I did think of 2 photon as a possibility, but I do not think it necessary
in this case.  And I do not have ready access to one.
  The secondary we are using is TRITC-goat-anti-rat.  Primary is a rat
monoclonal.  The tissue is mouse spinotrapezius.  The muscle is about 3-4
fibers thick in our working area.  The vessels that we need most to image
are in the center (of course).
  The point concerning the absorption of the myoglobin is one that I
considered, but had discounted.  Again, the brightness of the label is more
than sufficient.  Nonetheless, we could try a secondary labeled with Cy5
(something in the far red) to get further away from the myoglobin
absorption.  This may be more important than I had previously thought.  I
have not used the far red dyes very often because they are so hard to see
through the oculars!  The machine can see well enough, but I find it
difficult to locate the structures of interest....

__________________
Roger Adamson, PhD
Human Physiology
University of California
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Davis CA USA
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