Fig 1: Validation of expression of representative genes using immunohistochemistry. Overviews of DAB-immunohistochemical staining of OCM, PVALB, FBXO32, and SNCG and the representative images of the apical and basal regions indicated by the box in (A–D) were showed. Red asterisks indicate IHCs and black asterisks indicate OHCs in all panels. OCM (A) was highly expressed in OHCs while PVALB (B) and FBXO32 (C) expressed higher in basal than apical IHCs. No different expression of SNCG (D) has been found. (E) Comparison of IOD of representative genes between apical and basal IHCs. ∗∗p < 0.01. Scale bars: 100 μm for leftmost image of (A–D), 20 μm for right representative images of apical and basal regions of (A–D).
Fig 2: Validation of expression of representative genes using immunofluorescence in C57BL/6J mice. The basilar membrane was stained with antibodies against PVALB, OCM, FBXO32, and SNCG (red) and filamentous actin (green) was labeled with phalloidin. The labeling of PVALB (A) and FBXO32 (D) was intense in basal IHCs and weak in apical IHCs along the cochlear longitudinal axis. While OCM was highly expressed in OHCs and negatively expressed in IHCs (B). Selective expression deletion of Sncg (white triangles) was initially found in basal IHCs (C). Scale bar, 20 μm for all images in panels (A–D).
Fig 3: Validation of expression of representative genes using Immunofluorescence in BALBc mice. The basilar membrane was stained with antibodies against PVALB, FBXO32, and SNCG (red) and filamentous actin (green) was labeled with phalloidin. The labeling of PVALB (A) and FBXO32 (C) was higher in basal IHCs than that in apical IHCs while no selectively express deletion of SNCG (B) was observed in basal IHCs. Scale bar, 20 μm for all images in panels (A–C).
Fig 4: USeqFISH application to NHPs: in situ AAV detection and integrative analysis of cell morphology and transcriptional profiles.a,b, We applied USeqFISH to brain tissue slices of marmoset (a) and rhesus macaque (b) to which our viruses were administered (eight pooled variants for the marmoset and AAV.CAP-Mac for the rhesus macaque) with probes against three endogenous genes (yellow: Pvalb; green: Sst; magenta: Vip) and the coding sequence of each viral genome (human frataxin for the marmoset and mNeonGreen for the rhesus macaque (cyan); FPs were quenched by proteinase K (ProK) treatment). The representative images show that USeqFISH is applicable to these two NHP species with species-specific probes. c, Schematic of procedure of vector-assisted spectral tracing (VAST) and subsequent USeqFISH profiling of the rhesus macaque brain. We systemically delivered a cocktail of three AAV.CAP-Mac viruses packaging mNeonGreen, mTurquoise2 or mRuby2 to an infant rhesus macaque and recovered the brain. This brain exhibited a variety of colors, coming from stochastic expression of the three FPs, allowing us to trace single-cell morphologies. We additionally labeled seven endogenous genes (Pvalb, Sst, Vip, Lamp5, Slc17a7, Crym and Nr4a2) using USeqFISH in the same tissue to identify transcriptionally defined cell types and their morphology. d, Representative image of integration of VAST and USeqFISH with seven cell marker genes in the rhesus macaque brain and examples of two cells (yellow outlined box: i; red outlined box: ii) identifying both cell type and morphology.
Fig 5: USeqFISH validation in NHP tissue.a, Virally labeled cells, expressing mTurquoise2 (pseudo-colored), in the rhesus macaque brain and USeqFISH labeling with probes targeting the sequence of mTurquoise2. b, We validated our probe design for the rhesus macaque by applying USeqFISH with Pvalb probes and post hoc IHC with Pvalb antibodies to the same tissue. Localization of Pvalb mRNA signal in cells labeled with Pvalb antibodies (the same approach used for mouse probe validation in Supplementary Fig. 3) supports the versatility of our probe design in both rodents and NHPs.
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