The Levy Library Blog

a, Illustration of 8–12 ss mouse embryo. Dotted lines indicate the estimated position of the sagittal tissue section shown in b; D, dorsal; V, ventral; R, right; L, left; A, anterior; P, posterior. b, Tile scan of a 20-µm sagittal section of three independently sampled 8–12 ss embryos stained with nuclear dye DAPI (white). Red boxes indicate the selected field of view (FOV) imaged using seqFISH. c, Illustration of the experimental overview for spatial transcriptomics using seqFISH for 351 selected genes in 16 sequential rounds of hybridization and 12 non-barcoded sequential smFISH hybridization rounds for 36 genes. For each targeted gene, 17–48 unique probes were used to capture the mRNA; UMAP, uniform manifold approximation and projection. d, Cell segmentation strategy using a combination of E-cadherin (E-cad), N-cadherin (N-cad), pan-cadherin (Pan-cad) and β-catenin antibody (AB; green) staining detected by an oligo-conjugated anti-mouse IgG secondary antibody (orange) that gets recognized by a tertiary probe sequence. The acrydite group (blue star) of the tertiary probe (blue) gets crosslinked into a hydrogel scaffold and stays in place even after protein removal during tissue clearing. The cell segmentation labeling can be read by a fluorophore-conjugated readout probe (red); AB1, antibody 1; AB2, antibody 2. e, Cell segmentation staining of a 10-µm thick transverse section of an E8.5 mouse embryo using the strategy introduced in d. Cell segmentation signal was used to generate a cell segmentation mask using Ilastik (right). This was repeated independently for all N = 3 embryos with similar results. f, Representative visualization of normalized log expression counts of 12 selected genes measured by seqFISH to validate performance. This experiment was repeated independently for all N = 3 embryos with similar results. g, Highly resolved ‘digital in situ’ of the cardiomyocyte marker titin (Ttn), Tbx5, Cdh5 and Dlk1, colored in red, cyan, green and orange, respectively. Dots represent individually detected mRNA spots, and the box represents an area that was magnified for better visualization. This experiment was repeated independently for all N = 3 embryos with similar results.