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Immunofluorescence - intensity and background?


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#1 leroybrown

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Posted 08 October 2010 - 01:12 AM

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

#2 bguyonnet

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Posted 08 October 2010 - 05:53 AM

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


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!

#3 bob1

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Posted 08 October 2010 - 02:30 PM

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).

#4 leroybrown

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Posted 09 October 2010 - 09:13 AM

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




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