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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.
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.
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 ....
All experiments involving animals were performed in accordance with Northwestern University Animal Studies committee.
1. Preparation prior to organotypic cerebellar slice cultures
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.......
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.......
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.
....Name | Company | Catalog Number | Comments |
Alexa Fluor 594 Donkey anti-Mouse IgG secondary antibody | ThermoFisher scientific | A21203 | |
Basal Medium Eagle (BME) | ThermoFisher scientific | 21010046 | |
Biosafety cabinet Class II, Type A2 | NuAire | NU-540-400 | |
Bovine serum albumin | Millipore Sigma | A2153 | |
Brush | |||
anti-Calbindin D-28K antibody (CB-955) | Abcam | ab82812 | |
CO2 Incubator | NuAire | NU-5700 | |
Corning Costar Flat Bottom 6-well Cell Culture Plates | Fisher Scientific | 07-200-83 | |
Coverslips, 22 x 50 mm | Fisher Scientific | 12-545-E | |
Dressing forceps, straight | Harvard Apparatus | 72-8949 | |
Double edge blades | Fisher Scientific | 50949411 | |
Ethanol 200 proof | Decon Labs, Inc | 2701 | |
Eye Scissors, straight | Harvard Apparatus | 72-8428 | |
Fine forceps | Fisher Scientific | 16-100-127 | |
L-Glutamine 100X | ThermoFisher scientific | 25030149 | |
Glucose solution | ThermoFisher scientific | A2494001 | |
Hanks' Balanced Salt Solution | ThermoFisher scientific | 14025092 | |
Hoechst 33342, Trihydrochloride, Trihydrate | Fisher Scientific | H21492 | |
Horse Serum, heat inactivated, New Zealand origin | ThermoFisher scientific | 26050088 | |
ImageJ | |||
McIlwain Tissue Chopper | Fisher Scientific | NC9914528 | |
Microprobes | Fisher Scientific | 08-850 | |
Millicell Cell Culture Inserts | Millipore Sigma | PICM0RG50 | |
Nalgene Rapid-Flow Sterile Disposable Filter Units with PES Membrane, 250 mL | ThermoFisher scientific | 168-0045 | |
Nikon A1R confocal laser microscope system | Nikon | ||
NIS-Elements Imaging Software | Nikon | ||
Paraformaldehyde | Acros Organics | 41678-0010 | |
Pasteur pipets | Fisher Scientific | 13-678-20D | |
Potassium Chloride | Fisher Scientific | BP366-500 | |
ProLong Gold Antifade Mountant | ThermoFisher scientific | P10144 | |
Operating Scissors, straight | Harvard Apparatus | 72-8403 | |
Orbital shaker Belly Dancer | IBI Scientific | BDRLS0003 | |
Prism 8 | GraphPad | ||
Superfrost Plus Microscope Slides | Fisher Scientific | 12-550-15 | |
Tissue Culture Dish, 60 mm w/ grip ring | Fisher Scientific | FB012921 | |
Tissue culture plate, 24 well | Falcon/Corning | 353047 | |
Transfer pipettes, sterile | ThermoFisher scientific | 13-711-21 | |
Triton X-100 | ThermoFisher scientific | BP151-500 |
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