Following cell seeding and filtration, the microfilters were placed into 24-well culture dishes to facilitate simultaneous immunolabeling of multiple microfilters (instead of glass microscope slides) and washed for 10 min in 1x tris buffered saline (TBS; Biocare Medical, Concord, California). The filters were rehydrated with washes in decreasing concentrations of alcohol (100%, 90%, and 70%) for 6 min each wash. To quench any endogenous perioxidase activity, the microfilters were washed in 0.03% + methanol for 20 min. Following the perioxidase quench, the microfilters were washed with deionized , and nonspecific reactivity with primary antibodies was blocked with buffer containing 5% normal goat serum, 1% BSA, and 0.3% Triton X-100 in 1x TBS for 30 min. Rabbit antihuman CK (; DAKO, Carpinteria, California) and mouse antihuman CD45 (Ready-to-use; DAKO, Carpinteria, California) were cocktailed together and incubated with the microfilters overnight at room temperature. The microfilters were washed in 1x TBS for 10 min, and then incubated with MACH 2 secondary antibody buffer (Biocare Medical, Concord, California), containing both conjugated goat antirabbit alkaline phosphatase (AP) and goat antimouse horseradish perioxidase (HRP) activity, for 30 min. Microfilters were washed in 1x TBS for 10 min, then incubated with 3’, 3’-diaminobenzidine (DAB; Biocare Medical, Concord, California), reactive with HRP, for 5 min to form a brown precipitate reporting antiCD45 reactivity. Microfilters were washed in deionized , and then incubated with Warp Red (Biocare Medical, Concord, California), reactive with AP, for 7 min to form a red precipitate reporting antiPan CK reactivity. Microfilters were washed with deionized , incubated with CAT hematoxylin (Biocare Medical, Concord, California) 3 min for nuclear visualization, Tacha’s Bluing Reagent (Biocare Medical, Concord, California) for 3 min, dehydrated with increasing concentrations of alcohol (70%, 90%, and 100%), washed with xylene, and coverslipped using mounting medium (Richard Allan Scientific, Waltham, Massachusetts). To verify antibody specificity, cytospin slides were prepared using a mixture of SKBR-3 and peripheral blood mononuclear cells (), enriched by gradient-based centrifugation from a normal donor, and used as positive controls.