Making reluctant antibodies work

From:"Turner, Scott"

Does anyone have any experience in getting monoclonal antibodies that just
won't stain to work?  I've run into a rather intractable problem with this
one monoclonal antibody in particular that I can't get to work even on
frozen tissue.  It is a novel antibody to an antigen for which we have other
monoclonals that do work in immunohistochemistry, but this particular clone
won't stain our known positive control tissue (5 micron sections cut from an
unfixed, snap-frozen OCT block of mouse brain, then fixed 10 min in
acetone).  We know that it does bind the antigen both in vitro and in vivo,
but it doesn't work in Western blotting or IHC.  There are only two
possibilities I can think of as to why this mAb doesn't work: 1) the epitope
it binds is not available for some 3-d structural reason or 2) the fixation
is messing something up.  Does anyone have any other explanation that might
help me work around this issue?  Might it be better to employ ethanol,
methanol, n-butanol or some other fixative?   Is there some treatment that
might make the epitope available (trypsin, pepsin, protenase K, etc) that
anyone has had any success with in similar situations?  Any suggestions
about ways in which I might be able to make this antibody work would be

Scott Turner
DNAX Research
Palo Alto, CA

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