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. 2022 Oct 10;19(11):1393–1402. doi: 10.1038/s41592-022-01604-1

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© The Author(s) 2022

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 5 — a, Workflow for performing Light-Seq on the rare TH⁺ AC subtype, DACs: (1) Mouse retinas were fixed, frozen and cryosectioned. (2) After in situ RT, sections were stained with an antibody targeting the TH protein to label DACs (orange). (3) Barcoding of TH⁻ ACs with FITC-barcode strands (Bar1) and TH⁺ DACs with Cy3-barcode strands (Bar2) was performed in two rounds of light-directed barcoding, guided by the antibody stain. (4) After barcoding, cDNAs were displaced for sequencing, leaving the sample intact for further stains on the same cells. b, Representative image (n = 5 replicates) of one section replicate, stained with anti-TH antibody (orange) and DAPI (blue) before barcoding. For each replicate, only four to eight individual TH⁺ DACs were identified and their cell bodies were barcoded with Bar2 (magenta), together representing 0.01–0.02% of all cells in each section, and ~300 TH⁻ ACs were barcoded with Bar1 (green). Scale bars are 200 µm. c, Differential expression analysis revealed 36 transcripts enriched in DACs (P_adj < 0.05; two-sided Wald test with Benjamini–Hochberg adjustment for multiple hypothesis testing; genes with log₂(fold change) > 1 are shown; see source data) for n = 5 technical replicates. *Marker genes selected for further validation (log₂(fold change) > 3 and P_adj < 0.05). d, Fluorescently labeled barcodes (Bar1, Bar2) reveal the location of barcoded cDNAs, relative to the TH IF. Scale bars are 10 µm (n = 5 replicates, each with 4–8 TH⁺ cells per section). e, After cDNAs were displaced and sequenced, the same intact sections were stained for a membrane label (WGA) and a known marker of DACs via IF (CARTPT, cyan), in addition to the original TH IF and DAPI labels. f, Markers with log₂(fold change) > 3 and P_adj < 0.05 were validated using TH IF and RNA-FISH in new samples. Nondifferential controls, Gad1 and Vsx2, were also detected to demonstrate FISH labeling in TH⁻ ACs and other retinal cells. Top row shows overlay of RNA detection with TH IF, and bottom row shows single RNA-FISH channel. Scale bars are 10 µm. Representative images of n = 3–4 section replicates per marker.

Source data