title: Sample 5
organism: Mus musculus
description: Protocols: Mouse heart tissues were immediately frozen in liquid nitrogen after OCT compound (Tissue-Tek) embedding. Ten-micrometer tissue cryosections were stained with hematoxylin and eosin (H&E) and the appropriate tissue regions were selected for further processing. Samples were integrated to identify shared spatial domains using STAligner. Frozen heart tissue samples were embedded in OCT compound (Tissue-Tek) and subsequently cryosectioned at 10 µm thickness using a Cryostar NX70 cryostat (Thermo Fisher Scientific). Tissue sections were immediately transferred onto pre-chilled Visium Tissue Optimization Slides (10X Genomics, PN-1000193) to determine the optimal permeabilization time. Following manufacturer recommendations (10X Genomics), optimization experiments established an ideal permeabilization window at either 12 or 18 minutes for efficient tissue digestion and RNA release. For spatial transcriptomics, tissue sections were placed onto Visium Spatial Gene Expression Slides (10X Genomics, PN-1000187) and processed according to the Visium Spatial Gene Expression User Guide. H&E staining was performed as suggested by 10X Genomics. Brightfield imaging of tissue sections was performed using a Fritz brightfield digital microscope (Precipoint). Image stitching and processing were conducted using NIS-Elements software (Nikon).