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