Regulation of prefrontal cortex myelination by the microbiota methods
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Materials and methods
GF and CON Swiss Webster breeding pairs were obtained from Taconic (Germantown, NY, USA). GF mice were housed in gnotobiotic isolators under a strict 12-h light/dark cycle in cages of 4-5 animals. On postnatal day 21, just after weaning, exGF mice were taken from isolators and housed next to CON mice. CON and exGF mice were housed in the standard animal unit, which allows for exGF mice to be exposed to environmental microbes resulting in colonization. CON mice are similarly housed under regulated conditions (temperature 20-21 °C, 55-60% humidity), under the same 12-h light/dark cycle and housed 4-5 per cage. Autoclaved, pelleted diets were the same for all animal groups (Special Diet Service, Essex, UK, product code 801010). Animals were culled at week 10 for follow-up experiments. All experiments were approved by the Ethics Committee of University C...
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F 1 -generation offspring GF, exGF and CON mice were culled in adulthood at 10 weeks. Brains were collected for quantitative real-time PCR (qRT-PCR), for protein analysis and for transmission electron microscopy. For full detailed experimental flow with relevant animal number per experiment, see.
Transmission electron microscopy
Transcranial perfusion was carried out on six male mice ( n =3 per group), with 4% paraformaldehyde followed by PFC dissection. Following post-fixation in osmium tetroxide and dehydration in ascending ethanol series followed by propylene oxide, the samples were embedded in Araldite resin (Agar Scientific, Essex, UK). For each specimen, semi-thin (0.5 µm) and thin (70-90 nm) sections were obtained from polymerized blocks using a Reichert-Jung Ultracut E ultramicrotome (Leica-Microsystems, Wetzlar, Germany). Semi-thin sections were stained with toluidine blue and examined using a light microscope. Thin sections from selected areas of the trimmed blocks were made and collected on formvar-coated copper grids (Agar Scientific). Thin sections were double contrasted with 2% uranyl acetate and Reynolds lead citrate stain, and examined using a Jeol 2000FXII transmission...
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Evidence quotes (3)
GF and CON Swiss Webster breeding pairs were obtained from Taconic (Germantown, NY, USA). GF mice were housed in gnotobiotic isolators under a strict 12-h light/dark cycle in cages of 4-5 animals. On postnatal day 21, just after weaning, exGF mice were taken from isolators and housed next to CON mice. CON and exGF mice were housed in the standard animal unit, which allows for exGF mice to be exposed to environmental microbes resulting in colonization. CON mice are similarly housed under regulated conditions (temperature 20-21 °C, 55-60% humidity), under the same 12-h light/dark cycle and housed 4-5 per cage. Autoclaved, pelleted diets were the same for all animal groups (Special Diet Service, Essex, UK, product code 801010). Animals were culled at week 10 for follow-up experiments. All experiments were approved by the Ethics Committee of University College Cork and the Irish Department of Health authorities.
F 1 -generation offspring GF, exGF and CON mice were culled in adulthood at 10 weeks. Brains were collected for quantitative real-time PCR (qRT-PCR), for protein analysis and for transmission electron microscopy. For full detailed experimental flow with relevant animal number per experiment, see.
Transcranial perfusion was carried out on six male mice ( n =3 per group), with 4% paraformaldehyde followed by PFC dissection. Following post-fixation in osmium tetroxide and dehydration in ascending ethanol series followed by propylene oxide, the samples were embedded in Araldite resin (Agar Scientific, Essex, UK). For each specimen, semi-thin (0.5 µm) and thin (70-90 nm) sections were obtained from polymerized blocks using a Reichert-Jung Ultracut E ultramicrotome (Leica-Microsystems, Wetzlar, Germany). Semi-thin sections were stained with toluidine blue and examined using a light microscope. Thin sections from selected areas of the trimmed blocks were made and collected on formvar-coated copper grids (Agar Scientific). Thin sections were double contrasted with 2% uranyl acetate and Reynolds lead citrate stain, and examined using a Jeol 2000FXII transmission electron microscope (JEOL, Peabody, MA, USA), operated at 80 kV. Electron micrographs were obtained of areas of interest with a Megaview-III digital camera and AnalySIS software (EMSIS, Münster, Germany). A minimum of 50 myelinated axons were measured per animal.
Machine-readable layer
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