Immunofluorescence - intensity and background? - what can affect the two? (Oct/08/2010 )

Hi,

I considering adding a quantitative analysis to some immunofluorescence images and was wondering if anyone knows what factors, apart from target protein abundance, can affect the intensity of the stain in immunofluorescence?

On a second note, how exactly is 'background staining' defined? How do you tell the difference between a low level of expression of the protein and just background staining?

Many Thanks

Leroy

leroybrown on Fri Oct 8 09:12:32 2010 said:

Hi,

The concentration of the first and secondary antibodies can affect your intensity of fluorescence. For your "background staining", I think your are talking about control, aren't you? If it is true, you need a slide with a control for the specificity of binding of your primary antibody like pre-immune serum, whole serum, IgG, your first antibody blocked with your protein of interest.. that depend on the type of primary antibody you are using and a slide to control the specificity of binding of your secondary which is your secondary alone without primary antibody.

I hope that my answers could help you,

Good luck!

Background is usually also very non-specfic looking - i.e. the whole cell lights up at a low level. Intensity of staining can also be altered by too much washing or too stringent washing (i.e. too much salt or detergent in the washes).

Thanks, that makes sense.

I think the staining may be genuine, but I may try adjusting the detergent content of my washes to see how it affects it the staining.

There's always more experiments to do than you first think!

Thanks again