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Background In situ RNA sequencing analysis of constant or variable gene regions enables the possibility to study T-cell and B-cell immunology and pathology in a spatial context. Combining it with spatial protein analysis gives further insights into the immune repertoire and allows the correlation of specific tumor-associated antigen receptors which could help find and harness antigen-specific clones for therapy.Methods We used a newly developed cutting-edge multiplex sequencing and imaging platform which allows in situ sequencing, protein detection along with DAPI and H&E staining on the same tissue section. The platform can process two flow cells placed in a fully temperature-controlled environment using a closed fluidic system. Five µm thick tonsil and tumor FFPE sections were transferred to slides, followed by deparaffinization and antigen retrieval. A panel of amplifiable and fillable probes targeting the conserved V and C regions of the immune receptors was used. The subcellular detection of both known RNA transcripts and unknown sequences was performed using a four-color based in situ RNA sequencing-by-synthesis approach. RNA sequencing was performed first and then combined with Miltenyi MICS antibodies to identify proteins (>40) on the same tissue section.Results Feasibility data has been generated on the multiplex sequencing and imaging platform. Concurrently and on the same tissue section, an extensive panel of antibodies combined with multiplexed imaging and analysis techniques, was used to characterize T-Cell and B-Cell receptors in their spatial context. We observed high diversity of clonotypes which were assigned to T and B cell subtypes and used to generate a clonotype map. We also further applied immune cells protein markers for segmentation and mapping in the complex tissue environment.Conclusions The multiplex sequencing and imaging platform enables comprehensive spatial and phenotypic characterization of constant and variable segments of mRNA using in situ sequencing-by-synthesis. The integration of RNA detection with multiplexed antibody staining offers a powerful approach. We demonstrated in situ sequencing and the capability of characterizing TCR/BCR transcript on FFPE tissue. This approach enables researchers to identify and analyze the diversity of T-Cell and B-Cell populations based on their unique receptor sequences and spatially mapping immune cell clonality directly on tissue.