Fig. 1

High-content analysis of microfluidic immunofluorescence (IF) and fluorescence in situ hybridization (FISH). a Sequential IF/FISH staining. (i) Immunostaining of cell pellet or tissue section via the microfluidic tissue processor (MTP) clamped onto the tissue-carrying glass slide to deliver the reagents in a highly-controlled fashion. (ii) In the IF protocol, human epidermal growth factor receptor 2 (HER2) and cytokeratin (CK) proteins are tagged with rabbit anti-human HER2 antibody and mouse anti-human CK antibody and detected using AF594-labeled goat anti-rabbit IgG antibody (Ab) and AF647-labeled goat anti-mouse IgG Ab, respectively. Nuclei are marked with DAPI. The whole slide is scanned using a low magnification objective (×20). The image of a cluster of cells is presented (HER2: red, CK: green, DAPI: blue). (iii) In an elution step, staining agents are removed from the slide using proteolytic enzymes. iv In the FISH protocol, the HER2 loci are labeled with fluorescent HER2 probes, the centromeres of chromosome 17 are labeled with fluorescent chromosome enumeration probes (CEP17), and the nuclei are marked with DAPI. An image of the same cells as in ii is shown (HER2 loci: red, CEP17: green, DAPI: blue). b Image-processing of the IF and FISH images obtained after the protocol from a. IF and FISH images, aligned using the common DAPI channel, are sequentially processed. (i) For IF analysis, clusters of cells are segmented into an individual cell or a smaller group of cells based on nuclear analysis from the DAPI and CK channels (in blue). The HER2 signal also defines the area (in red) in which the mean HER2 and CK intensities for each cell are measured. (ii) For FISH analysis, nuclei (in blue) define the area where HER2 (outline in red) and CEP17 (outline in green) signals for each cell are scored. The HER2 copy number of each cluster is annotated by a number in red. c Data analysis pipeline. The cell HER2 expression given by IF is merged with the cell HER2 amplification obtained from FISH, followed by a filtering step that selects the cells of interest. For each sample, scores for HER2 protein overexpression and HER2 gene amplification based on the analysis of all the cells are obtained. Intra-tumoral heterogeneity analysis using spatial association analysis is also performed. Scale bars: 10 μm