non specific binding of FITC to erythrocytes

[Elena Ronander]

Hi,

I´m working with FISH on paraformaldehyde-fixed cells. The cells are labelled with a DIG-labeled RNA probe, subsequently labeled with an anti-DIG-HRP and then finally detected with FITC that reacts with the HRP. I get high unspecific binding to my erythrocytes without using the probe i.e on control slides where I´ve added only the FITC. I know that FITC is negatively charged and will bind positively charged molecules, but does anyone know how I can efficiently block/treat my slides in order to get rid of the unspecific binding ?

Regards,

Elena

[jiajia1987]

I am experiencing unspecific binding of my FITC to my streptavidin-coated microspheres too. Basically, I have streptavidin-coated microspheres to which biotinylated DNA and biotinylated anti-HA antibodies bind to. The biotinylated DNA actually has a sequence of HA at the 3' end, so after in vitro translation, peptides with HA tags are produced and they bind to the anti-HA antibodies on the beads. I detect these proteins using a peptide that is conjugated to FITC. During my analysis with flow cytometry, I realized that I get varying signals.

And in my negative control, there is supposed to be no DNA at all so no proteins are produced and no peptide-FITC should bind. But I get fluorescence signals in the negative controls and this indicates unspecific binding.

Been stumped at finding a way to get rid of the unspecific binding, though the only thing for now I can think of is to perform more stringent washings in between each step.