Optical Clearing of Fixed Brain Samples Using SeeDB

Meng‐Tsen Ke1, Takeshi Imai2

1 Graduate School of Biostudies, Kyoto University, Kyoto, 2 PRESTO, Japan Science and Technology Agency, Saitama
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 2.22
DOI:  10.1002/0471142301.ns0222s66
Online Posting Date:  January, 2014
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Abstract

Large‐scale three‐dimensional fluorescence imaging is essential for comprehensive and quantitative understanding of neuronal circuitry. We describe a water‐based optical clearing agent, SeeDB, which clears fixed brain samples in a few days leaving many types of fluorescent dyes unquenched, including fluorescent proteins and lipophilic neuronal tracers. This method maintains a constant sample volume during the clearing procedure, an important factor to keep cellular morphology intact. After optical clearing with SeeDB, we can reach a depth of >1000 µm with confocal microscopy. When combined with two‐photon microscopy, SeeDB allows us to image fixed mouse brains at millimeter‐scale level. Curr. Protoc. Neurosci. 66:2.22.1‐2.22.19. © 2014 by John Wiley & Sons, Inc.

Keywords: optical clearing; fluorescent imaging; two‐photon microscopy

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Standard SeeDB
  • Basic Protocol 2: SeeDB37
  • Alternate Protocol 1: SeeDB37ht
  • Alternate Protocol 2: SeeDBp
  • Support Protocol 1: Imaging Chamber Preparation
  • Support Protocol 2: Whole‐Mount Immunostaining
  • Support Protocol 3: Restoration for Long‐Term Storage and Histochemistry
  • Support Protocol 4: Neuronal Tracing by Lipophilic Dyes
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Standard SeeDB

  Materials
  • Mice
  • 4% (w/v) paraformaldehyde (PFA) in PBS
  • Phosphate‐buffered saline (PBS; )
  • 1% (w/v) low‐melting‐point agarose (e.g., Life Technologies) in PBS
  • 20%, 40%, 60%, 80%, and 100% (w/v) D(−)‐fructose solutions (see reciperecipes)
  • SeeDB (80.2% w/w fructose solution; see recipe)
  • 50‐ml conical centrifuge tube (for whole‐ and hemi‐brain samples; e.g., BD Falcon)
  • Culture dish (35 mm or 60 mm diameter; e.g., BD Falcon; for slice preparation and extremely fragile samples)
  • Tube rotator (recommended for clearing large samples; Fig. A)
  • Seesaw shaker (for fragile samples; Fig. B)
  • Air incubator (optional)
  • Moria perforated spoon (15‐mm head, e.g., Fine Science Tools, cat. no. 10370‐18, Fig. A)
  • Ring forceps (3 mm head, e.g., Fine Science Tools, cat. no. 11103‐09, Fig. A)
  • Additional reagents and equipment for mouse anesthesia ( ) and transcardial perfusion (unit ; optional)

Basic Protocol 2: SeeDB37

  Additional Materials (also see protocol 1)
  • SeeDB37 (84.4% w/w fructose solution; see recipe)

Alternate Protocol 1: SeeDB37ht

  Additional Materials (also see protocol 1)
  • SeeDB37 (84.4% w/w fructose solution)

Alternate Protocol 2: SeeDBp

  Additional Materials (also see protocol 1)
  • 20%, 40%, 60%, and 80% (w/v) D(–)‐fructose solutions (see reciperecipes, but prepare in in 0.1× PBS instead of water; see for PBS)

Support Protocol 1: Imaging Chamber Preparation

  Materials
  • Sample
  • SeeDB or SeeDB37 (see recipes) that was equilibrated with the sample (i.e., used in one of the preceding protocols)
  • Superglue
  • Appropriate immersion solution
  • Silicone rubber sheet (1‐6 mm in thick, e.g., Togawa Rubber, cat. no. K‐125)
  • Utility knife
  • Coverslips (24 mm × 60 mm for small preparation and slices; 50 mm × 70 mm for whole‐mount samples; e.g., Matsunami, cat. no. C050701)
  • Plastic bacterial Petri dish (100‐mm diameter; used to make a home‐made glass‐bottom dish); 100‐mm diameter glass‐bottom dish is also available from MatTek (glass diameter 30 mm, cat. no. P100G‐1.5‐30‐F).
  • Thermo Plate (e.g., Tokai Hit, http://www.tokaihit.com/; optional, only for samples cleared with SeeDB37 or SeeDB37ht)

Support Protocol 2: Whole‐Mount Immunostaining

  Materials
  • 4% (w/v) paraformaldehyde (PFA) in PBS (see for PBS)
  • 1% (v/v) Triton X‐100 in PBS (see for PBS)
  • Blocking solution (see recipe)
  • Primary and secondary antibodies
  • 20%, 40%, 60%, 80%, and 100% (w/v) D(−)‐fructose solutions (see reciperecipes)
  • Seesaw shaker
  • Multi‐well culture dishes (12 or 24 wells)
  • Additional reagents and equipment for serial incubation of samples with fructose solutions (see protocol 1)

Support Protocol 3: Restoration for Long‐Term Storage and Histochemistry

  Materials
  • 20%, 40%, 60%, 80%, and 100% (w/v) D(−)‐fructose solutions (see reciperecipes)
  • Phosphate‐buffered saline (PBS; )
  • Additional reagents and equipment for serial incubation of samples with fructose solutions (see protocol 1)

Support Protocol 4: Neuronal Tracing by Lipophilic Dyes

  Materials
  • 2% and 4% paraformaldehyde (PFA) in PBS (see for PBS)
  • DiIC 18(3) crystals (Invitrogen, cat. no. D282)
  • Scalpel
  • Aluminum foil
  • Additional reagents and equipment for serial incubation of samples with fructose solutions (see protocol 1)
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Figures

Videos

Literature Cited

   Chung, K. , Wallace, J. , Kim, S.Y. , Kalyanasundaram, S. , Andalman, A.S. , Davidson, T.J. , Mirzabekov, J.J. , Zalocusky, K.A. , Mattis, J. , Denisin, A.K. , Pak, S. , Bernstein, H. , Ramakrishnan, C. , Grosenick, L. , Gradinaru, V. , and Deisseroth, K. 2013. Structural and molecular interrogation of intact biological systems. Nature 497:332‐337.
   Dodt, H.U. , Leischner, U. , Schierloh, A. , Jahrling, N. , Mauch, C.P. , Deininger, K. , Deussing, J.M. , Eder, M. , Zieglgansberger, W. , and Becker, K. 2007. Ultramicroscopy: Three‐dimensional visualization of neuronal networks in the whole mouse brain. Nat. Methods 4:331‐336.
   Erturk, A. , Becker, K. , Jahrling, N. , Mauch, C.P. , Hojer, C.D. , Egen, J.G. , Hellal, F. , Bradke, F. , Sheng, M. , and Dodt, H.U. 2012. Three‐dimensional imaging of solvent‐cleared organs using 3DISCO. Nat. Protoc. 7:1983‐1995.
   Gonzalez‐Bellido, P.T. and Wardill, T.J. 2012. Labeling and confocal imaging of neurons in thick invertebrate tissue samples. Cold Spring Harb. Protoc. 2012. http://dx.doi.org/10.1101/pdb.prot069625.
   Hama, H. , Kurokawa, H. , Kawano, H. , Ando, R. , Shimogori, T. , Noda, H. , Fukami, K. , Sakaue‐Sawano, A. , and Miyawaki, A. 2011. Scale: A chemical approach for fluorescence imaging and reconstruction of transparent mouse brain. Nat. Neurosci. 14:1481‐1488.
   Helmchen, F. and Denk, W. 2005. Deep tissue two‐photon microscopy. Nat. Methods 2:932‐940.
   Ke, M.T. , Fujimoto, S. , and Imai, T. 2013. SeeDB: A simple and morphology‐preserving optical clearing agent for neuronal circuit reconstruction. Nat. Neurosci. 16:1154‐1161.
   Ragan, T. , Kadiri, L.R. , Venkataraju, K.U. , Bahlmann, K. , Sutin, J. , Taranda, J. , Arganda‐Carreras, I. , Kim, Y. , Seung, H.S. , and Osten, P. 2012. Serial two‐photon tomography for automated ex vivo mouse brain imaging. Nat. Methods 9:255‐258.
   Staudt, T. , Lang, M.C. , Medda, R. , Engelhardt, J. , and Hell, S.W. 2007. 2,2′‐thiodiethanol: A new water soluble mounting medium for high resolution optical microscopy. Microsc. Res. Tech. 70:1‐9.
   Tsai, P.S. , Kaufhold, J.P. , Blinder, P. , Friedman, B. , Drew, P.J. , Karten, H.J. , Lyden, P.D. , and Kleinfeld, D. 2009. Correlations of neuronal and microvascular densities in murine cortex revealed by direct counting and colocalization of nuclei and vessels. J. Neurosci. 29:14553‐14570.
Key References
   Ke et al., 2013. See above.
  This is the first report of the SeeDB protocol. Using optical clearing with SeeDB, this study also describes three‐dimensional layouts of callosal axons and mitral cell dendrites at single‐fiber resolution.
Internet Resources
   https://sites.google.com/site/seedbresources/
  SeeDB Resources provides updated information from the authors.
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