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Subsequently, according to the method described in the previous study ( 2.7 Immunofluorescence (IF) After transcardial perfusion in mice, brain tissues were harvested and pretreated with gradient dehydration in 20% and 30% sucrose solutions at 4C (to reduce ice crystal damage), then embedded in optimal cutting temperature compound and sectioned for 20 m thickness using cryostat

The levels of positive staining were quantified as the mean fluorescence intensity using ImageJ software [21]