antibody titration works like a bell curve.  you should see staining at a
weak concentration which increases in intensity until it becomes so
concentrated staining will disappear completely (over saturated).  this
is normal.  antibodies can be too concentrated, staining does not always
get better the more concentrated you make the antibody.  incubation time
is a different matter all together.  staining does usually get more
intense the longer you incubate with a weak or properly concentrated
antibody.
patsy

Jane Radford wrote:

> Dear Histonetters,
>
> I have a researcher working in our lab that is obtaining unusual
> results with his immunostaining.
> The researcher is using a rabbit anti human TRAIL (H257) antibody on
> human melanoma (intransit dermal metastasis) tissue. He has just
> competed a dilution series of the primary antibody (20, 10 and 5
> ug/mL) and found that the highest concentration (20ug/mL) actually
> resulted in no staining at all.  The other two dilutions gave a
> moderate level (10ug/mL) and very faint level (5ug/mL)  of staining
> which is what I would have expected. I would have logically expected
> the 20ug/mL concentration to have resulted in more intense staining.
>
> He made serial dilutions of the primary antibody so we know it must
> be in the solution and other than forgetting to apply the primary
> antibody or other components to the slide in question (which he
> assured me he didn't!) could there be a reason such as inhibition of
> binding of the primary antibody due to it being present in too high a
> concentration, or some other explanation?
> Thank you in advance for your help,
>
> Jane Radford
>
> Jane Radford
> Histopathology Laboratory Manager
> Department of Pathology
> University of Sydney
> NSW Australia
> Ph: 61 2 9351 6152
> Fax: 61 2 9351 3429
> janer@pathology.usyd.edu.au