Fig. 3.

Optimization of blocking strategy to decrease background signal in cancer cell-spiked human bone marrow. a Comparison of different blocking reagents in primary antibody unstained samples. Goat anti-rabbit AlexaFluor 488 (AF488) and goat anti-mouse AF647 secondary antibodies were applied. Images display merged signal from each fluorescence channel. Blocking reagents used were a: 10% goat serum; b: 10% human serum; c: 5% BSA; d: 10% goat serum with 5% BSA; e: 10% human serum with 5% BSA; f: Protein Block Serum Free; g: SuperBlock™; h: Image-iT™ FX Signal Enhancer; i: BlockAid™. b Effect of TrueBlack™ on background signal due to autofluorescence. Samples were stained with pan cytokeratin and white blood cell markers. Autofluorescent signal reduction is most evident in the AF555 channel (where no primary or secondary antibody was applied) and in the AF488 channel (stained for pan cytokeratin). Filled white arrowheads point to true positive staining. Scale bar = 50 μm