Whole‐Mount Immunohistochemistry of the Brain

Se‐Hoon Kim1, Priscilla Che2, Seung‐Hyuk Chung2, Debora Doorn2, Monica Hoy2, Matt Larouche2, Hassan Marzban2, Justyna Sarna2, Sepehr Zahedi2, Richard Hawkes2

1 Konyang University, Chungnam, 2 The University of Calgary, Alberta
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 2.10
DOI:  10.1002/0471142301.ns0210s36
Online Posting Date:  August, 2006
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The gross anatomical distribution of an antigen is typically mapped using a combination of serial sectioning, immunocytochemistry, and three‐dimensional reconstruction. This is a tedious and time‐consuming procedure, which introduces an array of potential alignment and differential shrinkage errors and requires considerable experience and specialized equipment. In particular, it is unsuited for routine screening applications. To circumvent these problems, this unit presents a routine whole‐mount immunocytochemistry protocol that can be used to map many antigenic distributions in the developing and adult brain. The technique can also be easily adapted to detect anterograde and retrograde transport tracers.

Keywords: cerebellum; development; pattern formation; antigen mapping; anterograde transport; retrograde transport

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Table of Contents

  • Basic Protocol 1: Whole‐Mount Fixation and Immunostaining of Brain Tissue
  • Alternate Protocol 1: Biotinylated Dextran Amine Injection in whole Mounts for Anterograde/Retrograde Axonal Projection Tracing
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Whole‐Mount Fixation and Immunostaining of Brain Tissue

  • Mice
  • Somnotol (MTC Pharmaceuticals)
  • 0.9% (w/v) saline, ice cold
  • 4% paraformaldehyde (PFA) in phosphate‐buffered saline (see recipe for PBS), ice cold
  • Dent's fixative (see recipe)
  • Dent's bleach (see recipe)
  • 100%, 50%, and 15% methanol
  • Phosphate‐buffered saline (PBS; see recipe)
  • 10 µg/ml proteinase K (>600 U/ml; Boehringer Mannheim) in PBS (see recipe)
  • 1 N HCl (optional)
  • PBST blocking solution (see recipe)
  • Primary antibody, e.g., anti‐zebrin II (Brochu et al., ), rabbit anti‐plCβ4 (Nakamura et al., ), or anti‐CaBP D28k (Swant, http://www.swant.com)
  • Horseradish peroxidase (HRP)–conjugated secondary antibody (e.g., Dako)
  • DAB solution (see recipe)
  • PBT (see recipe)
  • Sodium azide (NaN 3)
  • 10%, 20%, and 30% sucrose (optional)
  • 27‐G needle and 1‐ml syringe (for i.p. anesthesia)
  • Dissecting tools: scissors, forceps, blades
  • Magnifying lamp (optional)
  • −80°C freezer
  • Additional reagents and equipment for anesthesia by injection ( appendix 4B), perfusion fixation (unit 1.1), and cryosectioning (unit 1.1; optional)
NOTE: Throughout the protocol, gentle rocking or shaking is necessary for proper reagent penetration; ineffective penetration results in patchy whole‐mount staining. Ensure that the tissue is completely covered in solution at all times to prevent drying out.NOTE: When changing fixing and staining solutions, remove and dispense the solutions rather than moving the tissue.

Alternate Protocol 1: Biotinylated Dextran Amine Injection in whole Mounts for Anterograde/Retrograde Axonal Projection Tracing

  • Biotinylated dextran amine (BDA; Molecular Probes)
  • 0.1% (v/v) Triton X‐100 in phosphate‐buffered saline (see recipe for PBS); store indefinitely at room temperature
  • Biotin‐avidin Vectastain ABC kit (Vector Laboratories)
  • Stereotaxic frame (Stoelting)
  • Drill (Fine Science Tools)
  • 0.001‐ml Hamilton syringe (Fisher Scientific) and 26s‐G needle
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Literature Cited

   Brochu, G., Maler, L., and Hawkes, R. 1990. Zebrin II: A polypeptide antigen expressed selectively by Purkinje cells reveals compartments in rat and fish cerebellum. J. Comp. Neurol. 291:538‐552.
   Davis, C.A. 1993. Whole‐mount immunohistochemistry. Methods Enzymol. 225:502‐516.
   Dent, J.A., Paulson, A.G., and Klymkowsky, M.W. 1989. Whole‐mount immunocytochemical analysis of the expression of the intermediate filament protein vimentin in Xenopus. Development 105:61‐74.
   Hawkes, R. 1997. An anatomical model of cerebellar modules. Prog. Brain Res. 114:39‐52.
   Hawkes, R. and Gravel, C. 1991. The modular cerebellum. Prog. Neurobiol. 36:309‐327.
   Hawkes, R., Brochu, G., Doré, L., Gravel, C., and Leclerc, N. 1992. Zebrins: Molecular markers of compartmentation in the cerebellum. In The Cerebellum Revisited (R. Llinás and C. Sotelo, eds.) pp. 22‐55. Springer‐Verlag, New York.
   Luqué, J.M., Adams, W.B., and Nicholls, J.G. 1998a. Procedures for whole‐mount immunohistochemistry and in situ hybridization of immature mammalian CNS. Brain Res. Protoc. 2:165‐173.
   Luqué, J.M., Biou, V., and Nicholls, J.G. 1998b. Three‐dimensional visualization of the distribution, growth, and regeneration of monoaminergic neurons in whole mounts of immature mammalian CNS. J. Comp. Neurol. 390:427‐438.
   Miller, M.W. and Nowakowski, R.S. 1988. Use of bromodeoxyuridine‐immunohistochemistry to examine the proliferation, migration and time of origin of cells in the central nervous system. Brain Res. 457:44‐52.
   Nakamura, M., Sato, K., Fukaya, M., Araishi, K., Aiba, A., Kano, M., and Watanabe, M. 2004. Signaling complex formation of phospholipase Cβ4 with metabotropic glutamate receptor type 1α and 1,4,5‐triphosphate receptor at the perisynapse and endoplasmic reticulum in the mouse brain. Eur. J. Neurosci. 20:2929‐2944.
   Sarna, J. and Hawkes, R. 2003. Patterned Purkinje cell death in the cerebellum. Prog. Neurobiol. 70:473‐507.
   Sillitoe, R.V. and Hawkes, R. 2002. Whole‐mount immunohistochemistry: A high throughput screen for patterning defects in the mouse cerebellum. J. Histochem. Cytochem. 50:235‐244.
   Sillitoe, R.V., Malz, C., Rockland, K., and Hawkes, R. 2004a. Antigenic compartmentation of the primate and scandentid cerebellum: A common topography of zebrin II in Macaca mulatta and Tupaia belangerie. J. Anatomy 204:257‐269.
   Sillitoe, R.V., Marzban, H., Larouche, M., Zahedi, S., Affanni, J., and Hawkes, R. 2004b. Antigenic conservation of the architecture of the anterior lobe vermis of the cerebellum across mammalian species. Prog. Brain Res. 148:283‐297.
Key References
   Sillitoe and Hawkes, 2002. See above.
  A detailed description of the whole‐mount immunocytochemistry procedure as applied to the adult mouse cerebellum.
   Sillitoe et al., 2004b. See above.
  Examples of the application of the whole‐mount immunocytochemistry procedure applied to the cerebella of 23 different species, ranging from fish to primates.
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