Fluorescence

PERMEABILISIATION:For antibody diffusion.usually: PBS + 0.1-0.5 % TritonX-100 (3-5 min)BLOCKING:Mostly used: BSA or Gelatine (0.5 – 1%); for many antibodies blocking does not increase stainingefficiency -> „may not help, but doesn‘t hurt either“-> coffee break.PRIMARY-AB:Like in Western-needs higher titer. Rule of Thumb: 10 times the concentration that worked in Western(Western 1:1000 IF 1:100).Incubation: Room-Temperature; 45-60 min; if weak: either o/n 4oC or 2-3 hrs. at 37oC.Double-Stainings: Mind species cross-reaction!WASHING:3-5 x PBS for 5-10 minSECONDARY AB:Species-and Isoform-Specificity as in western; fluorophore-coupled.Termed INDIRECT Immunofluorescence (iIF) . In case the primary antibody is directly coupled: DirectImmunofluorescence (dIF) -> is more specific, though usually weaker since one antigen can only bind 1-2 antibodies, whereas secondary antibodies amplify the signal up to factors of 10.Incubation: Room Temperature; 45-60 min; if weak: either o/n 4oC or 2-3 hrs. at 37oC.Double-Stainings: Mind fluorophore cross-emission!After 2nd washing step : chance for DNA-counterstaining with DAPI, Hoechst (violet) or Propidium-Iodine (Red; broad; also stains RNA-> RNAse treatment recommended) or other DNA-binding dyes(SYPRO, YOPRO, TOTO – some cell-permeable, some not).