In situ hybridization strategy to visualize M-opsin and L-opsin mRNA.

(A) Identification of HEK293 cells using Hoechst (light gray) in the experiment in Fig 1I and 1J. M-opsin (blue) and L-opsin (pink). Blue arrow indicates a cell expressing M-opsin mRNA only. Pink arrows indicate cells expressing L-opsin mRNA only. Purple arrow indicates a cell expressing both M-opsin mRNA and L-opsin mRNA. Black arrow indicates an untransfected cell. Note that the Hoechst signal is reduced in cells that express M-opsin mRNA and/or L-opsin mRNA, likely due to the colorimetric signal blocking the fluorescent signal. (B) Identification of retinal layers using Hoechst (light gray) in the experiment in Fig 1K and 1L. M-opsin (blue) and L-opsin (pink). No cones co-expressed M-opsin mRNA and L-opsin mRNA. ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer. (C) Quantification of M-opsin mRNA-expressing, L-opsin mRNA-expressing, or M+L-opsin mRNA-expressing cells that express M/L-opsin protein for the experiments conducted in HEK293 cells in Fig 1I and 1J and adult human retina in Fig 1K and 1L.

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