ICC Negative Controls? - Immunocytochemistry (Jun/10/2007 )

Hi everyone,
I am doing Immunocytochemistry and I need to have some neagtive controls for controling for the specificity of antibody staining.
I am working with cell transfected with plasmids encoding a fusion protein.
I can use antibodies either specific to the protein or to the tag attached t it.

My question is:
What are the best negative controls for these experiment?

If using antibody against FLAG tag for eg.
Should my negative control be non-transfected cells stained with primary (anti-FLAG) and secondary antibodies?
or should it be transfected cells but staining only either with primary or only with ssecondary antibody?

I hope I was clear.
Thanks

You could stain untransfected cells with either the FLAG antibody or the protien specific antibody.

You could also use only the secondary antibody in the transfected cells.

to be a true negative control you should treat it exactly the same as your positive and test slides and still be confident it will come up negative (ie i'd use primary and secondary)

dom

Hi macedo,

it depends on the things you want to show...

1. To show that your transfected cells are transfected (and that your primary antibody stains the tagged protein in the transfecetd cells specifically) you stain untransfected cells using both, the primary and the secondary antibody. You should only get background or no signal in the untransfected cells...

2. To show that your secondary antibody interacts specifically with the primary one you use only the secondary antibody - here you expect to get no signal.
You may use untransfected or the transfected cells... as you like. When you use both in comparison you can say that your transfection doesn't change anything in reference to the binding of the second antibody (of course you can only say that when both show no staining ).

3. You also should stain a control of the cells containing the empty vector to show that your specific signals in the transfected cells is no signal because of the vector itself. Usually you get a signal somewhere in the cell because there is expression of the tag... but it shouldn't be in the region you expect your tagged protein to be.

Greetings,
Chakchel

Hi Chakchel
Thank you for your very clear reply,
I decided to use two negatives controls (option 1 and 2). Is more work but then I will be absolutely sure how specific is my antibody and if is not so specific I can dilute it more next time.

Thanks