User Ratings

Easy to Follow Achieved Expected Results Overall Rating Add your comments

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

¹Konyang University, Chungnam, South Korea
²The University of Calgary, Alberta, Canada

Publication Name:

Current Protocols in Neuroscience

Unit Number:

Unit 2.10

DOI:

10.1002/0471142301.ns0210s36

Online Posting Date:

August, 2006

GO TO THE FULL TEXT:

PDF or HTML at Wiley Online Library

Are you the author of this protocol? Login or register and return to this page.

Abstract

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

GO TO THE FULL PROTOCOL:

PDF or HTML at Wiley Online Library

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

GO TO THE FULL PROTOCOL:

PDF or HTML at Wiley Online Library

Materials

Basic Protocol: Whole-Mount Fixation and Immunostaining of Brain Tissue

Materials

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., 1990), rabbit anti-plC4 (Nakamura et al., 2004), 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 (NaN₃)
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: Biotinylated Dextran Amine Injection in whole Mounts for Anterograde/Retrograde Axonal Projection Tracing

Additional Materials (also see Basic Protocol)

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

Looking for materials? Suppliers Guide

GO TO THE FULL PROTOCOL:

PDF or HTML at Wiley Online Library

Figures

Figure 2.10.1

Whole-mount peroxidase immunocytochemical staining of cerebellum. (A) Adult Niemann-Pick type C mouse cerebellum (BALB/c npc^nih) stained using anti-calbindin, a marker of cerebellar Purkinje cells. The striped appearance is due to the selective patterned loss of Purkinje cells in this mutant (Sarna and Hawkes, 2003). (B) Anti-calbindin staining in adult nervous (nr/nr) mutant mouse. As in A, there is patterned Purkinje cell loss, but this time it is primarily the opposite set of cells to those stained in A that has been lost (reviewed in Sarna and Hawkes, 2003). (C) and (D) Immunoperoxidase staining using anti–zebrin II (Brochu et al., 1990) in an adult ground squirrel (C) (Spermophilus richardsonii) and a normal adult mouse (D) cerebellum. Zebrin II/aldolase C is selectively expressed by a Purkinje cell subset, revealing an intrinsic array of stripes. (E) Normal mouse cerebellum immunoperoxidase stained using anti–phospholipase C4 (Nakamura et al., 2004), which is expressed in the complementary subset of Purkinje cells to those expressing zebrin II (compare panels D and E). (F) Chick embryo at E14, immunoperoxidase stained for calbindin. At this age, calbindin expression reveals a stereotyped array of Purkinje cell stripes. (G) Newborn mouse cerebellum stained for calbindin. At this age, as in the chick embryo (F), calbindin expression reveals a stereotyped array of Purkinje cell stripes. (H) 5-Bromodeoxyuridine (BrdU) birthdating in the mouse. The dam was injected with BrdU at E11 and the cerebellum of the offspring was stained in whole mount at postnatal day 2 using anti-BrdU. The pattern of reaction product reveals birthdate-related stripes of Purkinje cells. Scale bars: Scale bars: A to F = 1 mm; G and H = 500 µm.

View Image
Figure 2.10.2

Retrograde labeling in cerebellum. (A) Mouse cerebellar Purkinje cells were retrogradely filled with BDA by making small injections into the intracerebellar white matter tracts in lobule VII, and detected using avidin-biotin immunocytochemistry. A stripe of Purkinje cell dendrites in lobule V, which does not extend into lobules III and IV, is indicated by the arrow. (B) Sagittal section through lobule V in A, restained to reveal BDA uptake in all layers of the cerebellar cortex. BDA-labeled Purkinje cells (arrow) are prominent in the molecular layer (ml), and their somata are seen in the Purkinje cell layer (pc). Labeling in the granular layer (gl) is due to mossy fiber axon terminals, and multiple afferent and efferent axons are immunoreactive in the white matter tracts (wm). Scale bar = 200 µm. (C) A BDA injection site in the vermis of lobule VI (black asterisk in center) and parallel fiber axon bundles extending laterally into the hemispheres (arrows). (D) A transverse section through the cerebellum shown in C. The granule cell axon trajectory can be traced from the somata in the granular layer (gl) through the Purkinje cell layer (pc) to the ascending axons traversing the molecular layer (ml; arrowheads), where they bifurcate to form a parallel fiber fascicle at the cerebellar surface (arrows). Scale bar = 100 µm. (E) Weak stripes of mossy fiber terminals (arrows) in the anterior lobe vermis can be seen following a BDA injection into the thoracic spinal cord. Scale bar = 500 µm. (F) The cerebellum in E was sectioned transversely and restained for BDA. Characteristic striped terminal fields (arrows) are prominent in the granular layer. Scale bar = 500 µm.

View Image

Literature Cited

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 C4 with metabotropic glutamate receptor type 1 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.