Browsing by Subject "in situ hybridization"

Prostate cancer is one of the most common cancers diagnosed in men. Somatic copy number alterations (SCNA), such as the deletion of PTEN or NKX3.1 and the amplification of MYC, have been associated with prostate cancer progression and could serve as potential biomarkers during diagnosis. One approach to utilize this information would involve screening a large number of prostate tissue sections for SCNAs and subsequently validating the findings using a secondary method. This process could enable more personalized treatment options for cancer patients. This thesis aimed to create a robust and reproducible workflow for SCNA identification. This was achieved by optimizing a chromogenic immunohistochemistry (IHC) protocol using immunostaining chambers and open-source 3D-printed laboratory hardware. The optimized protocol was then transferred to an automated liquid handling robot, and a panel of three antibodies for PTEN, NKX3.1, and MYC was developed for SCNA screening with IHC. Additionally, a chromogenic in situ hybridization (CISH) protocol was optimized to validate the results of the IHC. The immunostaining chambers required a lower antibody dilution to perform comparably to the manual IHC stainings. The automated protocol using the liquid handling robot produced high-quality stains with optimized dilutions. The optimized CISH protocol successfully identified the presence of the target gene, but unclear signals and merging of the signals obstructed detailed analysis. While complete deletion of PTEN was detectable, determining the number of gene copies per cell proved challenging due to signal variability and sample-dependent problems. Further optimization of the CISH protocol or development of an automated analysis workflow tailored to address these challenges is needed for more accurate analysis.