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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

Organotypic slice cultures are a powerful tool to study neurodevelopmental or degenerative/regenerative processes. Here, we describe a protocol that models the neurodevelopmental death of Purkinje cells in mouse cerebellar slice cultures. This method may benefit research in neuroprotective drug discovery.

Abstract

Organotypic slice culture is a powerful in vitro model that mimicks in vivo conditions more closely than dissociated primary cell cultures. In early postnatal development, cerebellar Purkinje cells are known to go through a vulnerable period, during which they undergo programmed cell death. Here, we provide a detailed protocol to perform mouse organotypic cerebellar slice culture during this critical time. The slices are further labeled to assess Purkinje cell survival and the efficacy of neuroprotective treatments. This method can be extremely valuable to screen for new neuroactive molecules.

Introduction

In vitro modeling is an essential tool in biomedical research. It allows investigators to study and tightly control specific mechanisms in restricted cell types, or in isolated systems/organs. Organotypic slice culture is a widely used in vitro technique, especially in the field of neuroscience1. The method was first established by Gähwiler, who cultured brain slices using the roller tube technique2, and later modified by Yamamoto et al., who introduced the use of a microporous membrane to perform cortical slice cultures3. Compared to primary cell cultures, organotypic slice cultures present ....

Protocol

All experiments involving animals were performed in accordance with Northwestern University Animal Studies committee.

1. Preparation prior to organotypic cerebellar slice cultures

  1. In a cell culture hood sprayed with 70% ethanol beforehand, prepare 200 mL of culture medium in a 250 mL bottle-top vacuum filter attached to a sterile bottle receiver. Add 100 mL of Basal Medium Eagle (BME), 50 mL of Hanks' Balanced Salt Solution (HBSS), 50 mL of heat-inactivated horse serum, 1 mL of.......

Representative Results

As shown in Figure 4, this protocol produces organotypic cerebellar slice cultures in which Purkinje cell survival can be assessed following the immunofluorescence and image acquisition steps. Purkinje cells were labeled with a combination of anti-Calbindin D-28K (dilution 1/200) and Alexa594 anti-mouse (dilution 1/300) antibodies. Image stitching was done automatically by the microscope acquisition software (NIS-Elements) to obtain a picture of the whole cerebellar slice. Purkinje cell numb.......

Discussion

Cerebellar slice culture is a powerful tool to study programmed Purkinje cell death during postnatal development. This technique can be used to rapidly screen candidate molecules for their neuroprotective potential. The main advantage is that the setup is simple and very cost effective, and only requires a modest investment in equipment (a vibratome can cost up to 3 times more than a tissue chopper). Moreover, 10 to 15 healthy slices can be generated from one mouse pup, allowing for different assays to be performed in pa.......

Acknowledgements

Imaging work was performed at the Northwestern University Center for Advanced Microscopy generously supported by NCI CCSG P30 CA060553 awarded to the Robert H Lurie Comprehensive Cancer Center. We thank Sean McDermott for his technical assistance and support, and Maya Epstein for the hand-drawn illustrations shown in Figure 1.

....

Materials

NameCompanyCatalog NumberComments
Alexa Fluor 594 Donkey anti-Mouse IgG secondary antibodyThermoFisher scientificA21203
Basal Medium Eagle (BME)ThermoFisher scientific21010046
Biosafety cabinet Class II, Type A2NuAireNU-540-400
Bovine serum albuminMillipore SigmaA2153
Brush
anti-Calbindin D-28K antibody (CB-955)Abcamab82812
CO2 IncubatorNuAireNU-5700
Corning Costar Flat Bottom 6-well Cell Culture PlatesFisher Scientific07-200-83
Coverslips, 22 x 50 mmFisher Scientific12-545-E
Dressing forceps, straightHarvard Apparatus72-8949
Double edge bladesFisher Scientific50949411
Ethanol 200 proofDecon Labs, Inc2701
Eye Scissors, straightHarvard Apparatus72-8428
Fine forcepsFisher Scientific16-100-127
L-Glutamine 100XThermoFisher scientific25030149
Glucose solutionThermoFisher scientificA2494001
Hanks' Balanced Salt SolutionThermoFisher scientific14025092
Hoechst 33342, Trihydrochloride, TrihydrateFisher ScientificH21492
Horse Serum, heat inactivated, New Zealand originThermoFisher scientific26050088
ImageJ
McIlwain Tissue ChopperFisher ScientificNC9914528
MicroprobesFisher Scientific08-850
Millicell Cell Culture InsertsMillipore SigmaPICM0RG50
Nalgene Rapid-Flow Sterile Disposable Filter Units with PES Membrane, 250 mLThermoFisher scientific168-0045
Nikon A1R confocal laser microscope systemNikon
NIS-Elements Imaging SoftwareNikon
ParaformaldehydeAcros Organics41678-0010
Pasteur pipetsFisher Scientific13-678-20D
Potassium ChlorideFisher ScientificBP366-500
ProLong Gold Antifade MountantThermoFisher scientificP10144
Operating Scissors, straightHarvard Apparatus72-8403
Orbital shaker Belly DancerIBI ScientificBDRLS0003
Prism 8GraphPad
Superfrost Plus Microscope SlidesFisher Scientific12-550-15
Tissue Culture Dish, 60 mm w/ grip ringFisher ScientificFB012921
Tissue culture plate, 24 wellFalcon/Corning353047
Transfer pipettes, sterileThermoFisher scientific13-711-21
Triton X-100ThermoFisher scientificBP151-500

References

  1. Humpel, C. Organotypic brain slice cultures: A review. Neuroscience. 305, 86-98 (2015).
  2. Gahwiler, B. H. Organotypic monolayer cultures of nervous tissue. Journal of Neuroscience Methods. 4 (4), 329-342 (1981).
  3. ....

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Purkinje CellOrganotypic Slice CultureCerebellar SlicesNeuroprotectionNeurodegenerative DiseasesTissue ArchitectureCell cell ConnectionsIn Vivo ConditionsPrimary Cell CultureCerebellumTissue HarvestingTissue ChopperParasagittal Sections

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