The HiSeq 2500 system has been obsolesced by new sequencers and Illumina has announcedthey will stop supporting the instrument and supplying the reagents in early 2023. The HiSeq was alegacy workhorse sequencing system, that had a large installation base around the world.The cost of acquiring this system is falling drastically because sequencing centers are decommissioningtheir HiSeqs and some are even available to purchase on eBay. The instrument itself is a powerfulsystem of scientific equipment that includes precise XYZ stages, fast imaging, and integrated fluidhandling. Several methods to spatially map multiplexed transcripts and/or proteins at subcellularresolution across tissue sections such as merFISH, STARmap, CODEX, 4i, and immuno-SABER have beenpublished using automated confocal microscopes with integrated fluidics that usually take days to run.We assessed the feasibility of repurposing a HiSeq 2500 instead of a dedicated confocal microscopesystem with integrated fluidics to perform spatial transcriptomics/immunofluorescence methods ontissue. We wrote software in Python, called PySeq2500, to control the stages, pumps, valves, optics, andcameras of the HiSeq. Custom flow cells were developed from off the shelf components to mount tissuesections and to interface with the HiSeq fluidics. Only minor modifications to the HiSeq stage were madeto accommodate the custom flow cell and to the fluidic plumbing to minimize dead volume. PySeq2500protocols were developed to automate various spatial transcriptomic/immunofluorescence methods onthe HiSeq 2500 and map targeted biomolecules across tissue sections. Adjacent tissue sections werealso mapped on a confocal microscope with integrated fluidics. The HiSeq and confocal systems werecompared in terms of data quality, performance, and ease-of-use. We show that HiSeqs that have beenopened up after being decommissioned for sequencing are still valuable systems that can performadvanced imaging based spatial mapping of biomolecules in tissue.