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. 2017 Nov 7;28(23):3360–3370. doi: 10.1091/mbc.E17-06-0408

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© 2017 Hendy et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 1: — Single mRNA transcripts detected by FISH in primary B-lymphocytes. (A) Sequence of the PD1 proximal promoter. Promoter elements are boxed and the mutations in each element indicated in red. (B) Detection and counting of single PD1-Cy3-labeled and CD19-Cy5-labeled mRNA transcripts in primary B-cells stimulated ex vivo. Left panel: Maximum-intensity projection of smFISH staining of B-cells; red and green spots indicate single PD1 and CD19 mRNAs, respectively. White arrow indicates CD19 transcription site with multiple nascent mRNAs. Right panel: Green squares identify individual mRNA molecules and circles identify transcription sites. (C) PD1/CD19 smFISH in a transgene-nonexpressing mouse. (D) smFISH with anti PD1-Cy3 and H2K^b-Cy5 probes in primary B-cells show nonoverlapping detection of endogenous and transgenic MHC class I mRNA. (E) Colocalization of smFISH spots for CD19 with two probe sets targeted to the 5′ and 3′ regions of the mRNA. (F) CD19 smFISH in B-cell stimulated culture. Image shows a CD19-expressing lymphocyte and a CD19-negative lymphocyte. (G) Bimodal distribution of CD19 transcripts indicates non–B-cells remaining in the population after stimulation. Black line indicates threshold for CD19 positive B-cells gated for downstream analysis. Scale bars represent 5 µm.