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Immunohistochemistry Mice were deeply anesthetized with sodium pentobarbital (300 mg per kg (body weight)) and perfused transcardially with 20 ml of prewarmed (37 C) saline, followed by 20 ml of prewarmed 4% paraformaldehyde in 0.16 M phosphate buffer (pH 7.27.4) and 50 ml of cold fixative