[Histonet] Re: Isotype background

[Johnson, Teri]

Hi Adam,

We have had the exact same experience you have sometimes when we're using Goat isotype and Rabbit isotype negative controls. Sometimes you can fiddle with the protocol and minimize some of the background, and sometimes you can't. Rabbit and goat antibodies/Igs are notoriously sticky. We use a non-serum protein blocker in our stain protocol and still have this happen.

The most ideal negative control would be non-immune serum from the animals that were immunized to produce the antibody. Since most places do not make that available, we have to purchase just normal animal species isotype control and hope for the best. This might explain why your antibody is not showing the background staining, but your negative control is.

Are you using any detergent in your wash buffers? You can try adding some Tween 20, 0.05% up to 0.1% or so and see if that cleans some of it up. Others might use Triton X-100, but we prefer to use Tween as it's a gentler detergent for slide-mounted samples.
How many rinses are you using? We rinse 3x between steps and that should be adequate.
You should be using a streptavidin block kit from Vector since you are using a conjugated streptavidin in your staining protocol.
Your use of donkey serum as a blocker and as an addition in your seconday antibody should block any Fc receptor staining (in theory).
Have you tried titering out the isotype control to the point of negativity? Do you ever get that?
What is your dilution of your secondary antibody? Streptavidin? Could those be titered out more to get better signal/noise ratio in your primary antibody treated sample?

Yes, you should try a null (no primary antibody - diluent only) control with your detection system on your sample and see if there is any background staining. But I would only use that to gauge the degree of background staining from your detection system in the absence of primary antibody. In most cases, unless there is some degree of endogenous staining, those slides are beautifully negative. Not ideal to use as a negative control other than demonstrating non-interference with the detection system.

I'm sorry I don't have any specific answers. Best I can do is sympathize and offer some possible things to look at.

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