Neuronal Death Assay to Evaluate Cell Death in Ex Vivo Epilepsy Model

Published: April 30, 2023

Abstract

Source: Carvalho, M., et al., A Model of Epileptogenesis in Rhinal Cortex-Hippocampus Organotypic Slice Cultures. J. Vis. Exp. (2021)

This video demonstrates the technique of assessing neuronal death in rhinal cortex-hippocampus organotypic slices. Neuronal death occurs via evolving epileptic seizures — resembling in vivo epilepsy — and is detected through dual-staining of the dead cells using propidium iodide and immunofluorescence.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board. 1. Preparation of rhinal cortex-hippocampus slices NOTE: The preparation of rhinal cortex-hippocampus slices uses P6-7 Sprague-Dawley rats. Culture setup and medium preparation On the day before the culture, prepare the required media and place them at 4 °C. Prepare d…

Representative Results

Figure 1: Detailed procedure for the preparation of rhinal cortex-hippocampus organotypic slices. (A) Remove the brain from the head and place it in ice-cold GBSS with the dorsal surface faced up. (B) Insert the forceps into the cerebellum. Open the brain through the midline and remove the excess tissue over the hippocampus. (C) With a spatula cut below the hippocampus, as indic…

Açıklamalar

The authors have nothing to disclose.

Materials

50 mL Centrifuge Tube, Conical Bottom Corning 430829
70% Ethanol Manuel Vieira, Lda UN1170
Anti-NeuN (rabbit) Werfen 16712943S Dilute at a ratio 1:500
B-27™ Supplement (50X), serum free Thermo Fisher Scientific 17504-044
Blades for scalpel handle Fine Science Tools 10011-00
Bovine Serum Albumin (BSA) NZYTech MB04602 5% BSA is used to dilute the primary antibodies. Add 0.5g BSA in 10 mL PBS.
Brain/Tissue Slice Chamber System Warner Instruments
Cell culture inserts, 30 mm, hydrophilic PTFE Millipore SAS PICM03050
Cold light source SCHOTT KL 300 LED
Confocal laser microscope Zeiss LSM 710
Conventional incubator Thermo Scientific Heraeus BB15, Function Line Set to 37 °C and 5% CO2
D(+)-Glucose monohydrate Merck Millipore 1.08342.1000
D-(+)-Glucose solution, 45% in water Sigma G8769
di-Sodium hydrogen phosphate dihydrate Merck Milipore 1.06580.1000
Dissecting microscope/magnifier MEIJI TECHNO CO. LTD 122285
Donkey anti-rabbit IgG (H+L) coupled to Alexa Fluor 488 Invitrogen A21206 Dilute at a ratio 1:500
Dumont #5 Fine Forceps Biologie Inox Fine Science Tools 11254-20
Dumont #5 Forceps Standard Inox Fine Science Tools 11251-20
Dumont #7 Forceps Standard Dumoxel Fine Science Tools 11271-30
Dumont Medical #7S Forceps Short Curve Inox Fine Science Tools 11273-22
Gentamycin stock solution, 50 mg/mL Thermo Fisher Scientific 15750-037
Gey’s Balanced Salt Solution (GBSS) Biological Industries 01-919-1A
Glass Electrodes Science Products GB150F-10 Round tips homemade
Glass Pasteur pipettes, 230 mm VWR International 612-1702
Hank’s Balanced Salt Solution (HBSS) Thermo Fisher Scientific 24020-091
Horse Serum, Heat Inactivated (HS)
Hydrochloric acid Thermo Fisher Scientific 26050-088
Hydrophobic Pen Merck Milipore 1.09057.1000
INCU-Line IL10 Dako S200230-2
Iris Spatula Curved Warner Instruments BSC-HT Haas Top
Labculture Class II Biological Safety Cabinet Fine Science Tools 10092-12
Lens Cleaning Paper HERASafe HS 12
L-Glutamine solution 200 mM (Q) TIFFEN
Micro tube 0.5 mL, PP Merck Millipore 1.05886.0500
Micro tube 1.5 mL, PP SARSTEDT 72,699
Micro tube 2.0 mL, PP SARSTEDT 72.690.001
Miroscope Cover Glasses, 24 mm x 60 mm Sutter Instrument MP-285
Nail polish Marienfeld 102242
Neurobasal-A Medium (NBA) Cliché
Opti-MEM® I Reduced-Serum Medium Thermo Fisher Scientific 10888-022
Paraformaldehyde, powder Thermo Fisher Scientific 31985-047
Peristaltic pump VWR Chemicals 2,87,94,295
Phosphate saline buffer (PBS) Gilson M312
Phosphate standard solutions, PO43- in water Homemade. PBS with 0.5% Tween-20 (PBS-T) is used to wash slices during the immunohistochemistry assay.
Pipette set BDH ARISTAR 452232C
Platinum 5 blades Gilson P2, P10, P20, P100, P200, P1000
Propidium iodide (PI) Sigma-Aldrich P5405-250g
Qualitative Filter Paper, Cellulose, Grade 1, 55 mm Sigma-Aldrich P4170-25MG Stock solution at 1 mg/mL in water.
Qualitative Filter Paper, Cellulose, Grade 1, 90 mm Whatman 1001-055 Medium retention 11µm
Scalpel handle Whatman 1001-090 Medium retention 11µm
Slip Tip Insulin Syringe without Needle 1 mL Fine Science Tools 91003-12
Student Scissors Straight SharpSharp 12cm Astro Med Inc GRASS Product Group S48 Stimulator
SuperFrost Plus™ Adhesion slides Fine Science Tools 91402-12
TC-Treated Sterile 60 x 15mm Tissue Culture Dish Thermo Fisher Scientific J1800AMNZ
TC-Treated Sterile 6-Wells Plates Corning CORN430166
Tissue Chopper MEDICAL SYSTEMS CORP. TC-102
Triton X-100 The Mickle Laboratory Engineering CO. LTD. MTC/2 Set to 350 μm
Tween-20 BDH 14630
Sigma P2287

Etiketler

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Bu Makaleden Alıntı Yapın
Neuronal Death Assay to Evaluate Cell Death in Ex Vivo Epilepsy Model. J. Vis. Exp. (Pending Publication), e21283, doi: (2023).

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