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. 2022 Sep 2;15:976349. doi: 10.3389/fnmol.2022.976349

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Copyright © 2022 Tsuneoka, Atsumi, Makanae, Yashiro and Funato.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Representative photomicrographs of two-round ISH-IHC. The first HCR visualized Drd1 (green, S41-SaraFluor488), Drd2 (yellow, S23-Atto550), and Vgat (magenta, S68-Cy5) mRNAs. The second HCR visualized Vglut2 (green, S45-SaraFluor488), Penk (yellow, A161-Atto550), and Pdyn (magenta, S85-Cy5) mRNAs. In the IHC, tyrosine hydroxylase, oxytocin-neurophysin, and nNos were visualized by Alexa488, Alexa568, and Alexa647 secondary antibodies, respectively. (A) Merged low-magnification images of mouse sections, including the preoptic area (POA) and striatum. Rectangles indicate the regions shown in (B,C). Scale bar: 200 μm. (B) Merged, sequential images of the first HCR, photobleached image, the second HCR, photobleached image, and IHC. Scale bar: 50 μm. (C) Separate high-magnification images of each molecule. Scale bar: 20 μm.