The AAV (BrainVTA Co., Ltd., Wuhan, Hubei, China) that overexpressed MORwildtype/MORY7.43A was rAAV2-EF1α-EGFP-WPRE-hGH polyA, AAV-PHP.eB vector containing MORwildtype/MORY7.43A insert of mice.
The heads of mice were firmly fixed by the thumb and index finger. A 25 µl tip microsyringe was used to slowly inject AAVs (5 µl) into the right cerebral lateral ventricle (at coordinates −1.0 mm mediolateral, −0.5 mm anteroposterior from Bregma; −2.0 mm dorsal–ventral from the skull) (Craft et al., 2005 (link)).
Twenty-one days following virus injection, mice were sacrificed through transcardial perfusion of deeply anesthetized mice using intraperitoneal-injected 1% sodium pentobarbital. Mice were perfused with 0.9% saline followed by 4% paraformaldehyde in 0.1 M PO4, after which whole-brain dissections were performed. Brains were postfixed overnight in 4% paraformaldehyde in 0.1 M PO4 followed by incubation for a minimum of 48 h in 30% sucrose in 0.1 M PO4 (Kofoed et al., 2021 (link)). Fifty-micrometer sections were cut on a sliding microtome with a freezing stage (Leica Biosystems, Buffalo Grove, IL, United States) (Rincon et al., 2018 (link)). Sections were collected in a 24-well plate filled with antifreeze (PBS: glycerol: ethylene glycol = 5:2:3) and stored in a refrigerator at −20°C and were scanned using Olympus VS.120.
The heads of mice were firmly fixed by the thumb and index finger. A 25 µl tip microsyringe was used to slowly inject AAVs (5 µl) into the right cerebral lateral ventricle (at coordinates −1.0 mm mediolateral, −0.5 mm anteroposterior from Bregma; −2.0 mm dorsal–ventral from the skull) (Craft et al., 2005 (link)).
Twenty-one days following virus injection, mice were sacrificed through transcardial perfusion of deeply anesthetized mice using intraperitoneal-injected 1% sodium pentobarbital. Mice were perfused with 0.9% saline followed by 4% paraformaldehyde in 0.1 M PO4, after which whole-brain dissections were performed. Brains were postfixed overnight in 4% paraformaldehyde in 0.1 M PO4 followed by incubation for a minimum of 48 h in 30% sucrose in 0.1 M PO4 (Kofoed et al., 2021 (link)). Fifty-micrometer sections were cut on a sliding microtome with a freezing stage (Leica Biosystems, Buffalo Grove, IL, United States) (Rincon et al., 2018 (link)). Sections were collected in a 24-well plate filled with antifreeze (PBS: glycerol: ethylene glycol = 5:2:3) and stored in a refrigerator at −20°C and were scanned using Olympus VS.120.
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