Unspecific signals in fluorescence staining of neutrophil extracellular traps

Dear all,

I am trying to stain RNA in neutrophil extracellular traps by using appropriate primary antibodies and corresponding secondary antibodies (polyclonal from chicken with Alexa Flour 594 or 647 tag). I am facing problems with unspecific signals. Even if I stain my NET samples with the secondary antibody only, I am achieving strong signals which are very similar to staining with primary and secondary antibodies. For blocking I am using pooled human serum for 1 h at RT after I permeabilize my cells with 0.1 % saponin for 15 min at RT. I have also tried blocking with BSA (5 % and 3 %) and permeabilization with 0.1 % Triton X 100 but the results are the same. I am using primary human blood neutrophils and my primary antibodies are mouse anti-human, my secondary antibodies are chicken anti-mouse. For imagaing I am using a Zeiss LSM800 Confocal microscope.
Does anybody know what the issue could be? Maybe I need a monoclonal secondary antibody or one from another species than chicken? Do you think blocking with another solution or longer incubation time could help? Maybe someone who works with neutrophils, too, faces similar problems? For NET induction I am using PMA, which is the standard NET inducer.

Thank you a lot for your help and best regards,
Francesca

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I don’t think you can do much more blocking than you did. What concentration of secondary are you using? You could try diluting the secondary, which may improve the ratio of specific to non-specific binding. I’d guess that you will have the best luck trying secondaries from different species (polyclonal), for example I usually use goat or donkey anti-X antibodies (from Jackson labs) for staining cultured cells. A different dye might also help, sometimes the non-specific interaction comes from the dye and not the antibody itself.