[Histonet] Re: staining of negative controls with IHC

From:"Johnson, Teri"

MaryAnn Dixon wrote:
"We are currently retrieving animal tissue in a pressure cooker with a
program of 125 degrees for 30 secs.  This protocol was taken from
biocare but I'm sure this is for human tissue. Most of the tissue is
retrieved with Biocare's Reveal (pH 6.0).  We are receiving some
background staining of our negative controls whereas if we did not
retrieve them, there is none.  We were staining for KI-67 which is a
nuclear stain. I am using a tonsil for the control. The negative control
showed some sporatic cytoplasmic staining on the Universal negative
control from biocare as well as deionized water. Since then we have
taken the temperature down to 115 degrees and increased the time to 25
minutes.  It seems to have decreased the cytoplasmic staining but there
is still some lingering.  Anyone out there have any ideas??? I'm sure
there is a better protocol for retrieving animal tissues. It has to be a
time and temperature thing!"

MaryAnn, you don't mention what type of animal you are staining, and
what your IHC protocol looks like.

I would never use a universal negative control  when doing
immunostaining on animals. Use only the Igs from the species the primary
antibody was made in. If you are using a monoclonal mouse KI-67, you
would need to use non-immune mouse IgGs of the same isotype of your
primary antibody. If your antibody is a rabbit polyclonal or monoclonal,
you'd need to use non-immune rabbit IgG. It's also important that you
match the Ig concentration fo the non-immune (negative) control to the
Ig concentration of your primary antibody. If you are using pre-diluted
antibodies, you may be using different Ig concentrations.

Try an additional run to see if your detection system is causing the
staining. Run a tonsil, and do the pressure cooker retrieval as usual,
however and omit the primary antibody, using only diluent or buffer in
the incubation. Then continue your IHC protocol as usual. If there is
still some background/non-specific staining, it's due to your detection
system (kit?) and not to the antibody or negative control reagent.

Additionally, I never use a universal reagent for detection. Making sure
your antibody reactions are appropriate are difficult enough without
throwing in additional animal species you don't need.

If you still have specific questions, don't hesitate to contact me
off-list and I'll be happy to take a look at your protocol and make some

Good luck!

Teri Johnson, HT(ASCP)QIHC
Managing Director Histology Facility
Stowers Institute for Medical Research
1000 E. 50th St.
Kansas City, MO 64110

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