[Histonet] Controls for Nickel enhanced DAB

From:"Pixley, Sarah \(pixleysk\)"

Dear Bob Nienhuis:

My preferred control (somewhat old fashioned and hard core now) for IHC
and antibody experiments is to use a solution that is as close to your
primary antibody as possible.   So if your primary antibody is
unprocessed rabbit serum, then normal rabbit serum is your best control.
Sera vary greatly, especially in their non-specific staining. So of
course the optimal control solution is the pre-immune sera from the
animal that you got your primary from, taken prior to injection of the
antigen!! But today it is extremely rare to be able to have that.  So
here are two other ways to ensure a good control.  First, do a protein
determination on both sera and make sure that you are using at least the
same protein concentration (or more) than you have in your primary.
Second, some rabbit sera just have endogenous background binding. I had
to throw one out one year because it was so much higher in background
than my primary antibody.  You may have to stain and try some to find
one that does not give too high a background. Is this completely kosher?
I rationalized it because the great variation between animal sera is
natural and some sera just do give background.

If your primary antibody is either a mouse monoclonal or a processed
primary antibody, then the place that you obtained your antibody can
help you find a good control. For the mouse monoclonal, ATCC often sells
specific antibodies or solutions that serve as controls for their
primaries. These might be, for example, culture supernatants from a
non-antibody producing cell line that was grown with the same culture
supernatant, and collected and processed similarly. If you have a
primary serum that is processed, the commercial companies will work with
you to find a similarly processed solution that does not have any
antibody in it that will bind to your tissue. I.e., I have used
antibodies against bacterial proteins from the same company, processed
in the same way. ETC.  These are much better controls than anything you
can come up with in your lab.

Removing primary is usually deceptively clean. It does NOT provide a
good background check!!!!

Another important thing to do in your case is to do a dilution curve of
the primary antibody. That will allow you to see if the background goes
down while specific staining stays. 

FYI, I used to use cobalt-nickel enhancement, but it was horrible to
work with. Too much precipitate. I now use a stock of 2% nickel sulfate,
NiSO4.6H2O: 0.9 g into 45 ml E-pure dd H2O = 2% solution. Keep in
Fridge. Dilute: 1/25 in your DAB solution to give a 0.08% solution.
Seems just as sensitive, gives a good blue-black color and less
precipitate in both stock and the DAB solution, although there still is

Best wishes.
Sarah Pixley

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