Re: Negative Controls Immunos

From:Patricia Bourne <>

Dear Donna,

Negative controls should be run on each block studied.
Processing and fixation will and can very from block
to block. At the very least one should use the
antibody diluent as a negative control.  The next best
would be non-immune serum from the same species as the
primary antibody.  Dako has both of these available at
a reasonable cost.  The dilution for the negatives
should be run at the same titer of your most
concentrated (strongest) antibody dilution.  Example:
if Keratin is run at 1-25 and is the most concentrated
antibody then your negative control is set at that
titer (1-25).  The theory is that if it doesn't stain
at the most concentrated titer is won't stain at
higher dilutions such as 1-100 or 1-300 etc.  The
other alternative is having matched controls for each
antibody Keratin 1-25 with negative control at 1-25 or
LCA 1-100 with the negative control at 1-100.  This
can lead into increased amounts of work and from
personal experience is unnecessary.  

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