3 replies to this topic

[suncherry]

I'm doing immunocytochemistry on THP-1 macrophage with Texas Red as second antibody, but there is strong signal under FITC light as well.Is is autofluroscent? What's the reason? How to avoid it?

Thanks.

[miBunny]

If you see a signal without using texas red, the cells are autoflourescing.  This may be happening but most likely,  what you are seeing is the texas red spilling into the green channel.  

You can try titering down the amount of secondary antibody (texas red conjugated) you are using.

[suncherry]

What's the concentration of Texas Red conjugated second antibody you usually use? I apprrciate your help. Thanks.

[miBunny]

Every secondary antibody will be different and it is always best to titer your secondary antibody.  TO do this, keep the amount of primary antibody constant and test a range of dilutions of your secondary.  Choose the lowest concentration that still gives you a good signal.