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Dear Colleagues,

Immunofluorescence can be manipulated just like any other technique.
What is important is to establish valid and stringent criteria and
sticking to them throughout your experiments. Then you will have a
basis to decide if there is more/less/no co-localization in response
to treatment. Using automations/software in a mindless manner will
not help; for example, I can get less than 5% co-localization between
A and B, but if it 90% is in the nucleolus then that is highly
significant no matter what the software decides.

Does the finding make biological sense? That is pre-eminent.
Conclusions should be supported by more than one method- one should
be able to reproduce protein binding and/or immunoprecipitation of
the complex. What I think is fraudulent is having so many "not nice"
images claimed as showing co-localization, that is merging an
essentially 'green' field with a 'red' one and showing 'yellow'
without any detail. That is the same as not washing your
immunoprecipitates and claiming that proteins A and B are in the same
complex.

Fred E. Indig, Ph.D.
Head, Confocal Imaging Unit

Research Resources Branch
National Institute on Aging/NIH
5600 Nathan Shock Dr.
Baltimore, MD  21224

Tel. 410-558-8173
Fax  410-558-8236
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