Spatial Analysis of Perivascular Adipose Tissue Gene Expression with SABER-FISH

Perivascular adipose tissue (PVAT) is a critical regulator of vascular homeostasis, modulating immune responses, and vessel contractility. This modulatory effect is primarily mediated by its secretome, with PVAT adipocytes being one of its main sources; however, the biology of these cells is poorly understood. PVAT's adipocyte populations are maintained by the differentiation of adipocyte progenitor cells, a stem cell population with multilineage potential. The potential of adipocyte progenitors to sustain a healthy PVAT underscores the need for precise characterization of these populations at both the spatial and molecular levels. High-resolution imaging of PVAT poses significant challenges due to the complex and heterogeneous composition of adipose tissue (adipocyte phenotypes, lipid content, and extracellular matrix composition). Conventional immunohistochemistry techniques are limited by autofluorescence, light scattering, and impaired antibody penetration. Alternatives, such as nucleic acid detection, have gained importance, including fluorescence in situ hybridization (FISH). Newer versions, such as SABER-FISH (signal amplification by exchange reaction-FISH), utilize DNA-based elongation of probes with primer exchange reaction to enhance their specificity and signal strength. This paper presents a methodology for applying SABER-FISH in PVAT. We address key technical limitations and provide strategies to enhance signal specificity and improve imaging analysis, thereby increasing the utility of FISH for investigating gene expression in this metabolically and immunologically active tissue.