ARTICLE |
Correspondence to: Richard Hawkes, Dept. of Cell Biology & Anatomy, Faculty of Medicine, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta T2N 4N1, Canada. E-mail: rhawkes@ucalgary.ca
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Summary |
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Large-scale mouse mutagenesis experiments now under way require appropriate screening methods. An important class of potential mutants comprises those with defects in the development of normal cerebellar patterning. Cerebellar defects are likely to be identified often because they typically result in ataxia. Immunohistochemistry (IHC) is commonly used to reveal cerebellar organization. In particular, the antigen zebrin II (=aldolase C), expressed by stripes of Purkinje cells, has been valuable in revealing cerebellar pattern abnormalities. The development of whole-mount procedures in Drosophila, chick, and Xenopus embryos allows complex patterns to be studied in situ while preserving the integrity of the structure. By combining procedures originally designed for embryonic and early postnatal tissue analyses, we have developed a whole-mount IHC protocol using anti-zebrin II, which reveals the complex topography of Purkinje cells in the adult mouse cerebellum. Furthermore, the procedure is effective with a number of other antigens and works well on both perfusion-fixed and immersion-fixed tissue. By use of this approach, normal adult murine cerebellar topography and patterning defects caused by mutation can be studied without the need for three-dimensional reconstruction.
(J Histochem Cytochem 50:235244, 2002)
Key Words: Purkinje cell, zebrin, mutagenesis, calbindin, lurcher, NPC, pattern formation
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Introduction |
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THE MAMMALIAN CEREBELLUM is an ideal model in which to explore pattern formation in the CNS. Although cerebellar histology appears uniform, functional, anatomic, and molecular studies have all revealed an elaborate topography. The cerebellum is organized around an array of transverse zones (reviewed in
Pattern formation is crucial to normal cerebellar function. If Purkinje cell stripe formation is disrupted, the consequences are abnormal topography and severe motor control problems [e.g., disabled (mdab1-1) (
With the advent of large-scale murine mutagenesis experiments, there is a need to rapidly screen for patterning defects in the cerebellum. However, although zebrin II expression is a sensitive way to detect abnormal Purkinje cell patterns, the three-dimensional (3D) reconstruction of cerebellar topography is far too slow for routine screening purposes. To address this problem, we have developed a whole-mount IHC staining procedure for the adult mouse cerebellum. The method is a combination of a staining technique developed for the embryonic and neonatal opossum brain (
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Materials and Methods |
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Mice
All animal procedures conformed to institutional regulations and the Guide to the Care and Use of Experimental Animals from the Canadian Council for Animal Care. CD1 mice were purchased from Charles River Laboratories (Montreal, PQ, Canada). Cerebellum-deficient folia mice (cdf:
Antibodies
Anti-zebrin II, a mouse monoclonal antibody (MAb) produced by immunization with a crude cerebellar homogenate from the weakly electric fish Apteronotus (
Mouse MAb10B5 was raised against neurospheres from the embryonic murine striatum (
Both anti-zebrin II and MAb10B5 were produced in our laboratory and used directly from spent hybridoma culture medium.
Mouse monoclonal anti-calbindin (anti-CaBP, IgG1 isotype: Swant, Bellinzona, Switzerland) was used diluted 1:500. In the adult mouse cerebellum, CaBP is expressed exclusively by Purkinje cells. All Purkinje cells are immunoreactive.
Horseradish peroxidase-conjugated rabbit anti-mouse immunoglobulins (Jackson ImmunoResearch Laboratory; West Grove PA) was diluted 1:200.
Fixation and Sectioning
For perfusion-fixation, mice were injected IP with 100 mg/kg somnotol. When the toe-pinch reflex was undetectable, the mice were transcardially perfused with ice-cold 0.9% saline until all saline flushing out of a laceration made in the right atrium was void of blood. Ice-cold 4% paraformaldehyde in 0.1 M PBS, pH 7.4 (Sigma; St Louis, MO) was then perfused for approximately 6 min to allow complete fixation of the cerebellum. The cerebellum was removed from the calvaria and immediately placed in ice-cold fixative without removal of the meninges. The tissue was allowed a further 2448 hr of postfixation at 4C. No difference in staining due to fixation time was seen. For immersion-fixation, mice were anesthetized as above and the brains removed from the calvaria. The cerebellum was then separated from the rest of the brain and immersion-fixed, ice-cold, for 2448 hr in two or three changes of 4% paraformaldehyde in PBS.
Whole-mount Immunohistochemistry
Throughout the protocol, gentle rocking or nutation is necessary for proper reagent penetration. Ensure that the tissue is completely covered in solution at all times to prevent drying out. We have used 2.0-ml cryotube vials (Nalge Nunc International; Naperville, IL) filled to 1.5 ml. Each cerebellum was processed separately.
Steps
1. After allowing the cerebellum to bathe in fixative for 2448 hr, it is postfixed in Dent's fixative [4 parts absolute methanol (MeOH), 1 part dimethysulfoxide (DMSO) (
2. Incubate the tissue in Dent's bleach [4 parts MeOH:1 part DMSO:1 part 30% hydrogen peroxide (H2O2)] until the tissue is completely white (8 hr) to block endogenous peroxidase activity.
3. Dehydrate the cerebellum twice for 30 min in 100% MeOH. This is important because large amounts of water trapped in the tissue may form ice crystals on subsequent freezing.
4. Subject the tissue to four or five cycles of freezing in 100% MeOH to -80C in a freezer and thawing to room temperature (RT) on the bench. The frozen tissue is brittle and care must be taken to avoid damage, especially while it thaws. Five cycles of freezing ensure that antigens in the deeper parts of the cerebellum are rendered accessible. However, we have successfully revealed zebrin II expression in Purkinje cell dendrites with only two cycles of freezing.
5. Incubate the tissue overnight in 100% MeOH at -80C to ensure good reagent penetration. The tissue can be stored for prolonged periods at this stage.
6. Rehydrate the tissue for 90 min each in 50% MeOH, 15% MeOH, and PBS at RT.
7. Enzymatically digest the tissue in 10 µg/ml proteinase K (>600 U/ml: Boehringer Mannheim, Laval, PQ, Canada) in PBS for 5 min at RT to permit free penetration of reagents.
8. Rinse the cerebellum three times for 10 min in PBS at RT.
9. Block nonspecific antibody binding by incubating the cerebellum in 2% non-fat skim milk powder, 0.1% Triton X-100 in PBS (PBSMT;
10. Incubate the tissue for 48 hr in primary antibody diluted in PBSMT containing 5% DMSO at 4C (to improve penetration).
11. Rinse the tissue in PBSMT two or three times for a period of 23 hr each at 4C.
12. Incubate the tissue overnight at 4C in secondary antibody diluted in PBSMT + 5% DMSO.
13. Once again rinse the tissue in PBSMT three or four times for 23 hr each at 4C, followed by a final overnight incubation at 4C.
14. Rinse the cerebellum in 0.2% bovine serum albumin, 0.1% Triton X-100 in PBS (PBT;
15. Peroxidase binding sites are revealed by incubating in a freshly-prepared 3,3'-diaminobenzidine (DAB; Sigma, St Louis, MO) solution consisting of 1 x 10 mg DAB tablet in 20 ml PBT and 10 µl 30% H2O2.
16. The peroxidase reaction can be stopped once the desired color intensity is reached by washing the cerebellum in PBT + 0.04% sodium azide. (Even though sodium azide is an effective inhibitor of bacterial and fungal growth, it should be avoided until this step because it is also an inhibitor of horseradish peroxidase.)
17. If clearing is desired, the tissue is dehydrated completely in sequential rinses of 30%, 50%, 80%, and 100% MeOH for 3060 min each at RT then transferred to 1 part benzyl alcohol in 2 parts benzyl benzoate for clearing (BABB). The clearing solution should be stored in glass or polypropylene containers because it readily dissolves polystyrene.
Section Immunocytochemistry
Cerebella to be sectioned were cryoprotected through graded sucrose solutions: 10% (2 hr), 20% (2 hr or until the cerebellum sank), and 30% (overnight) at 4C. The tissue was then frozen in OCT embedding compound (VWR; Mississauga, ONT, Canada). Serial sections were cut at 50 µm in the transverse plane and mounted on gelatin-coated slides. Sections for IHC staining were processed by using slide-mounted (IHC) and mounted in DPX (BDH Chemicals; Toronto, ONT, Canada). Briefly, slide-mounted sections were rinsed two times for 5 min each in PBS, incubated in 1% H2O2 for 10 min, and then rinsed three times for 5 min each in PBS. The sections were then incubated in 10% normal goat serum (NGS) in PBS and 0.5% Triton X-100 for 2 hr at RT. The tissue was then left in primary antibodies diluted in 10% NGS, PBS, and 0.5% Triton X-100 overnight at 4C. After rinsing three times in PBS for 5 min each, the tissue was incubated in secondary antibody diluted in NGS, PBS, and 0.5% Triton X-100 for 2 hr at RT. Antibody concentrations were the same as those used for whole-mount IHC. A freshly prepared DAB solution was used to visualize antibody binding, as for the whole mounts. Sections from cerebella previously immunoperoxidase-stained in whole mount were mounted directly.
Whole-mount photomicrographs were captured with a SPOT digital camera (Diagnostic Instruments; Sterling Heights, MI) mounted on a Zeiss Stemi SV6 microscope. Cerebella were photographed immersed in PBT, with incident illumination. Montages were assembled in Adobe Photoshop 4.0 (Tucson, AZ). The images were cropped and corrected for brightness and contrast but not otherwise manipulated.
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Results |
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Zebrin II Expression in Normal Mouse Cerebellum
The vermis of the cerebellar cortex is constructed around four transverse zones: the anterior zone (AZ; lobules IV), central zone (CZ;
lobules VIVII), posterior zone (PZ;
lobules VIIVIII), and nodular zone (NZ;
lobules IXX), each of which is further subdivided into parasagittal stripes (e.g.,
500 µm laterally to either side. More lateral still, a broader, weakly immunoreactive P3+ is seen. [Zebrin-immunoreactive stripes are numbered from P1+ medially to P7+ laterally: zebrin-negative (P-) stripes are numbered according to the immediately medial P+ stripe (
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The AZ extends caudally to the dorsal aspect of the primary fissure, where it interdigitates with stripes derived from the CZ (
Zebrin II Expression in the Cerebellum Deficient Folia Mutant
To demonstrate the utility of zebrin II whole-mount IHC on a mutant background, we have stained cerebella from the ataxic cerebellum deficient folia (cdf/cdf) mouse (
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Other Cerebellar Antigens
Calbindin (CaBP) expression in the cerebellum is restricted entirely to Purkinje cells [rat (
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Fig 4A4D show anti-CaBP immunoreactivity in the Lc/+ cerebellum at postnatal day (P)43, at which age the majority of Purkinje cells have degenerated (
Antibody Penetration in Whole Mount-immunostained Cerebella
In whole-mount staining it appears likely that the density and thickness of the adult cerebellar tissue poses a major barrier to antibody penetration. To determine the extent of antibody penetration, cerebella stained in whole mount were cryostat-sectioned and examined (Fig 5). Zebrin II and CaBP are Purkinje cell antigens and therefore might be detected even if antibody penetration into the tissue is limited. Indeed, the standard recipe described above favors the detection of antigens in the molecular layer of the cerebellar cortex. However, by increasing the incubation period in the primary antibody from 48 to 96 hr (Step 10) and in the secondary antibody from overnight to 2 days (Step 12), antigens can be detected in all layers of the cerebellum. As an example, we have used the anti-granule cell antibody MAb10B5 (
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Discussion |
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The whole-mount IHC protocol described here has several advantages for routine screening for cerebellar patterning defects. By use of anti-zebrin II, a reliable high-resolution topographical map can readily be demonstrated. Previous studies have shown zebrin II IHC to be a powerful tool. Its expression reflects the fundamental molecular architecture of the cerebellum and is sensitive to genetic manipulations of cerebellar development [e.g., Lc/+ (
Whole-mount IHC combines the advantages of zebrin II IHC with the ability to see the pattern without sectioning and 3-D reconstruction. Not only is it much more rapid but it is at least as reliable and requires far less effort. As a result, whole-mount IHC can easily be automated to generate a routine screen for cerebellar patterning mutants. We have also successfully used whole-mount fluorescence IHC (unpublished data), which suggests that double-label protocols could be developed straightforwardly. Because the protocol works well with immersion-fixed tissue (e.g., Fig 2A), it is also unnecessary to include a time-consuming transcardiac perfusion. In addition, zebrin II IHC is compatible with many fixatives and, if necessary, the cerebella can be subsequently sectioned for histological analysis.
Before sectioning the tissue, it is also possible to clear the stained cerebella to reveal patterning deeper within the tissue. The clearing agent that is chosen is usually determined by matching its refractive index as closely as possible with that of the tissue. For embryos, BABB appears to work best after sequential methanol dehydration (reviewed in
This protocol was developed on the basis of IHC principles already widely used. The major departure from previously published whole-mount protocols (
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Acknowledgments |
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Supported by grants from the Canadian Institutes of Health Research and the Ara Parseghian Foundation for Medical Research (RH).
We thank Robert Stack and Matthew Larouche for husbandry and genotyping of the NPCnih mice, Estrella Gonzales for technical assistance, Dr Michael Vogel for the gift of Lc/+ cerebella, and Drs Len Eisenman (Philadelphia) and Cairine Logan (Calgary) for discussions and advice.
Received for publication July 9, 2001; accepted August 29, 2001.
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