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Fig. 1 | Biological Procedures Online

Fig. 1

From: Optimization of Immunofluorescent Detection of Bone Marrow Disseminated Tumor Cells

Fig. 1

Identification of sources contributing to background staining in cancer cell-spiked human bone marrow. a Demonstration of true pan cytokeratin+ and white blood cell- (CK + WBC-) putative cancer cell and CK + WBC+ putative WBC using the basic protocol. b Autofluorescence and non-specific secondary antibody binding are evident after rehydration of the slides and secondary antibody incubation, respectively. After all steps are performed, CK staining is detected by AlexaFluor 488 (AF488, green) and CD45 by AF647 (magenta). All images capture signal from all channels, although there is no staining in the AF555 channel. c Autofluorescence in primary unstained bone marrow fixed with 4% PFA for 30 min or 10 min. Filled white arrowheads point to true positive staining. Dashed circles indicate background staining on representative cells due to autofluorescence, while open solid arrowheads indicate false staining due to non-specific binding of secondary antibody. Scale bar = 20 μm for merge zoom image, 50 μm for merged and respective individual channel images in the remainder of the figure

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