Neuroscience
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Preparation and Immunostaining of Myelinating Organotypic Cerebellar Slice Cultures
Chapters
Summary March 20th, 2019
Here we present a method to prepare organotypic slice cultures from mouse cerebellum and myelin sheath staining by immunohistochemistry suitable for investigating mechanisms of myelination and remyelination in the central nervous system.
Transcript
This method allows to study the basic mechanism of myelination and remyelination at the cellular and tissular level. This technique is at the crossroad between primary cultures and in vivo approaches. It is a fast and accessible model with the main advantage of preserving the tissue architecture.
Demonstrating the procedure will be Melina Thetiot, a postdoctoral researcher and Remi Ronzano, a PhD student from my laboratory. To begin, insert a small scissors gently into the foramen magnum and cut the skull by making one lateral incision towards the side, and then cut all around the head skull. To retrieve the dorsal part of the skull, use a fine, straight forceps to carefully lift the dorsal part of the skull.
Then carefully introduce the forceps between the ventral skull and the brain. Gently flip out the brain and cut the optic and trigeminal nerves with small scissors. Next, turn the head or the dorsal part of the skull upside down just above a 60 millimeter cell culture dish containing ice cold dissection medium to help the brain to drop by gravity.
Using fine forceps, orientate the brain with the dorsal side facing up and the ventral side lying down. Under the binocular microscope, use the fine, straight forceps to immobilize the brain on the forebrain side. Then, separate the hind brain from the rest of the brain.
Cut the cerebellar peduncles underneath the cerebellum to separate the cerebellum from the rest of the hind brain. Once the cerebellum is isolated, use fine, straight forceps to carefully tear away the meninges. Hold the cerebellum gently with the fine, curved forceps and place it with the dorsal side up onto the plastic platform, perpendicularly to the chopper razor blade.
Next, with a sterile, thin end pipette tip attached to a one milliliter pipette, aspirate any access of dissection medium around the cerebellum. Then, with a tissue chopper, slice 300 micrometer thick saggital sections of the cerebellum. Next, add a drop of dissection medium onto the sliced cerebellum gently.
Then, with a wide boar pipette tip attached to the one milliliter pipette, slowly aspirate the sliced cerebellum and transfer it back into the 60 millimeter cell culture dish containing ice cold dissection medium. Next, use two fine, straight forceps to separate individual slices. Then, with a wide boar pipette tip attached to the one milliliter pipette, transfer up to four selected slices from the vermis, along with some dissection medium onto one culture insert of a six well plate.
Remove any access of dissection medium around the slices using a thin end pipette tip. For demyelination, remove all culture medium below the culture inserts after six days in vitro, and replace it with one milliliter per well of the pre-warmed, fresh culture medium containing 0.5 milligram per milliliter LPC. Incubate for 15 to 17 hours at 37 degrees Celsius in 5%carbon dioxide environment.
After incubation, wash the inserts by placing them in a 25 milliliter Petri dish containing one milliliter of pre-warmed culture medium. Then, immediately transfer the culture inserts in a new, six well plate, containing fresh, pre-warmed culture medium. To begin immunohistochemistry, first use forceps to lift culture inserts and remove the culture medium beneath them.
Then, add two milliliters of 4%PFA in 1x PBS, ph 7.4, onto the membrane inserts to fix the cerebellar slices. After 30 minutes, wash the slices three times with two milliliters of 1x PBS for 10 minutes each wash. Next, under the binocular microscope, using a 25x to 30x magnification, use a scalpel or a brush to gently detach the slices from the membrane inserts.
Then, use a brush to transfer the floating slices into the wells of a four well plate, containing 1x PBS to limit antibody consumption. Then, aspirate the PBS from each well and incubate the slices in pre-cooled 100%ethanol at minus 20 degrees Celsius for 15 to 20 minutes. After incubation, aspirate the 100%ethanol and wash the slices briefly with 1x PBS.
Then, wash them two times at room temperature with 1x PBS for 10 minutes each wash. To block non-specific antibody fixation sites, aspirate the PBS and incubate the slices in a solution containing 1x PBS, 5%NGS, and 0.3%non ionic detergent at room temperature for 30 to 45 minutes. Next, add primary antibodies diluted in the blocking solution and incubate the slices at four degrees Celsius overnight.
After overnight incubation, wash the slices three times with 1x PBS for 10 minutes each wash. Then add the secondary antibodies, diluted in the blocking solution, at one to 500 dilution ratio and incubate the slices at room temperature in the dark for three hours. After incubation with the secondary antibody, wash the slices three times in 1x PBS in the dark for 10 minutes each wash.
Next, under a binocular microscope, place 100 microleters of 1x PBS on the slide and with a brush, transfer the slices into the PBS and flatten them on the slide. Remove any access of PBS. Finally, place a drop of mounting medium directly onto the glass cover slip and gently cover the slices.
Myelin immunostainings in organotypic cerebellar slices, obtained from post natal days 9 to 10 C57 black six wild type mice were observed at 11 days in vitro with nodes of ronviae enriched in voltage gated sodium channels, flanked by the paranodal axoglial junction domain. Same results were observed for PLP-GFP transgenic mice. Myelination of the parkengy cells is mostly achieved after one week in culture.
LPC treatment fully demyelinates the slices, which remyelinate spontaneously and are fully myelinated six days post demyelination. This procedure should take 25 minute maximum. It is really the most critical point.
Organotypic slice culture are suitable for live imaging study of myelination dynamics. It can also be used for targeting drug screening experiments. This technique allows an easy, quantitative approach to study myelination and remyelination processes.
Experimenters are to follow the basic safety procedures applying to animal welfare, excessive and sharp myelination.
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