Immunostaining of metastatic breast cancer patient blood samples. Representative images of positive ERα and HER2 staining in clinical samples. Row A, from left to right: DAPI counterstain (fluorescent blue), cytokeratins 8, 18, and 19 (CK) stained with Phycoerythrin (red), estrogen receptor (ERα) stained with AlexaFluor 488 (green), and a composite of all channels. This patient sample (no. 4, see Table 3) was collected prior to initiation of therapy, illustrating two clustered ERα+ CTCs, adjacent to a solitary leukocyte located in the lower left corner. This patient was diagnosed with an ERα+ metastasis. Row B, from left to right: DAPI counterstain (fluorescent blue), HER2 stained with Liquid Permanent Red (red). This patient sample (no. 1, see Table 3) was obtained following 6 months of chemotherapy, illustrating HER2+ CTCs identified by combination of fluorescence and bright-field microscopy. This patient was diagnosed with a HER2− primary tumor and HER2− metastasis.