In breast cancer, 15-20% of cases are reported with overexpression of human epidermal growth factor receptor 2 (HER2) that causes rapid cancer progression and poor prognosis. Fortunately, HER2-targeted therapy using specific antibodies such as Trastuzumab is effective for treating these cases. In situ hybridization (ISH) is a standard technique used for HER2 assessment. This technique locates the HER2 gene by using complementary DNA probes. Two main ISH methods are fluorescence in situ hybridization (FISH) and chromogenic in situ hybridization (CISH). However, both FISH and CISH are expensive and laborious. Moreover, they can only be assessed in a small area of the tissue and thus are prone to errors in case of HER2 intra-tumoral heterogeneity (ITH). This thesis aims to improve HER2 assessment in breast cancer through various techniques related to microfluidics. The main component of our technology is a microfluidic tissue processor. This micro-fabricated chip is clamped to a microscope slide carrying a human tissue slice, creating a chamber that accommodates the tissue and delivering reagents to stain it. Five applications of this microfluidic technology to breast cancer diagnostics are presented in the three chapters of this thesis. In chapter 1, we present a microfluidic FISH protocol for HER2 gene assessment using a standard FISH probe and demonstrate that, when applying an oscillatory flow within the chip, hybridization efficiency is increased thanks to molecular replenishment. This back-and-forth motion of the diluted FISH probe inside a thin chamber above the tissue slide is the principle of microfluidic-assistance for ISH. We thereby succeed in drastically reducing the experimental time from 2 days to 1, and the amount of the expensive probe used per test by a factor of 10. We highlight the performance and reliability of microfluidic-assistance FISH by comparing the FISH scores obtained by this method to standard FISH technique scores using several clinical tissue samples. The principle of microfluidic assistance in FISH is also applicable to other types of ISH probes, including fast FISH based on Ethylene Carbonate and CISH. In chapter 2, we describe a new microfluidic method allowing the quantification of HER2 expression levels from formalin-fixed breast cancer tissues. After partial extraction of proteins from the tissue slide, the extract is routed to an antibody microarray for HER2 titration by fluorescence. HER2-negative and positive samples can be distinguished using this simple test, and the obtained results agree with the FISH scores. In chapter 3, we establish a method allowing high content, cell-by-cell analysis of both protein overexpression and gene amplification using successive microfluidic immunofluorescence (IF) and FISH staining combined with image processing. We demonstrate that by using high-content automatic analysis, the HER2 status of the sample can be precisely assessed using both a quantitative IF technique based on HER2 and cytokeratin protein quantification and automatic scoring of HER2 loci and centromere of chromosome 17 signals in a FISH image. Furthermore, this method characterizes HER2 ITH quantitatively. In particular, heterogeneous clusters and individual cells are visualized in a reconstructed map of the tissue. We conclude that high-content IF/FISH analysis is a powerful tool that can assist clinical diagnostics in the future.