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Neuro Imaging

開催日 2014/9/11
時間 10:00 - 11:00
会場 Room H(304)
Chairperson(s) 喜多村 和郎 / Kazuo Kitamura (東京大学大学院医学系研究科 神経生理学 / Department of Neurophysiology, Graduate School of Medicine, The University of Tokyo, Japan)
今井 猛 / Takeshi Imai (理化学研究所 発生・再生科学総合研究センター(理研CDB) / RIKEN Center for Developmental Biology, Japan)

Correlative histochemistry: a quick and simple method for whole-mount immunohistochemistry and optical clearing of in vivo-imaged neurons

  • O1-H-2-1
  • 今井 猛 / Takeshi Imai:1,2 柯 孟岑 / Meng-Tsen Ke:1 
  • 1:理研CDB / RIKEN CDB, Kobe, Japan 2:JSTさきがけ / JST PRESTO, Japan 

In vivo calcium imaging has facilitated our understanding of information coding by ensembles of neurons in the brain. However, in many situations, it is not easy to analyze the correlation between neuronal response profiles and neuronal subtypes defined by specific marker proteins. Existing methods for whole-mount immunohistochemistry have limitations for depth and/or require complicated procedures for efficient antibody penetration and staining. Here we demonstrate that a simple lipid removal procedure (16-24 hours) facilitates antibody penetration for whole-mount immunostaining of adult mouse brains with minimal sample deformation. We also developed a new one-step, water-based, and non-toxic optical clearing agent, named SeeT (See Through), which can more quickly (several hours) and efficiently clear brain samples than existing agents. Combining these two procedures, we could stain and visualize epitopes (e.g., GFP, GAD67, and parvalbumin) located down to 1mm depth from the surface of the brain and reconstruct them in 3D. Our method is suitable for immunohistochemical analyses of neurons previously characterized by GCaMP calcium imaging in vivo (termed Correlative histochemistry). Correlative histochemistry should facilitate our understanding of in vivo neuronal responses, in relation to neuronal subtypes, and the mesoscopic connectome of the same animal.

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