Enhanced Spatial Transcriptomics Analysis of Mouse Lung Tissues Revealed Cell-Specific Gene Expression Changes Associated with Pulmonary Hypertension

We developed a method that utilizes floating mounting of thin sections of fixed frozen mouse lung tissue onto Xenium slides for the fluorescent in situ hybridization (FISH) and imaging–based spatial transcriptomics analysis of gene expression using the Xenium platform provided by 10X Genomics. Spatial transcriptomics techniques provide a comprehensive view by merging gene expression data with spatial context within their native tissue architecture. However, the Xenium pipeline has been validated only for formalin-fixed paraffin-embedded (FFPE) and fresh frozen sections by 10X Genomics. Notably, many researchers prefer paraformaldehyde-fixed cryosections for immunohistochemistry and in situ hybridization. In our study, we assessed the compatibility of standard fixed frozen mouse lung sections with the Xenium protocol. Our findings reveal that these sections not only align well with the Xenium platform but also offer superb imaging and gene expression quantification, even with limited number of genes in the Xenium panel. This protocol can serve as a valuable resource for preparing various tissues where FFPE and fresh frozen samples present challenges. Total four mice lung sections were included. Two sections were from two Egln1Tie2Cre (KO) mice, and two sections were from littermate controls. Mouse lung was first fixed by 4% PFA through transcardial perfusion, dissected out, and further post-fixed in 4% PFA overnight at 4 °C. The lung was then cryoprotected, embedded in OCT compound, and sectioned on a cryostat into 10um thickness. Xenium slide is fully mounted with sections, and then take a note on tissue sections and layouts, and record the metadata information for each section (e.g. from which mouse, age, sex, genotypes etc). The slide is then stored in a slide mailer and kept at -80 °C up to three weeks. The predesigned 379-gene mouse panel of probes provided by 10x Genomics was used to conduct the high-resolution Xenium in Situ Analysis experiment.