Quantification of GSK3 in PV Interneurons

Immunohistochemistry was performed as previously described [16 (link)]. Briefly, sections containing auditory cortex were double-immunostained with rabbit anti-GSK3α (1:200, G08-63R-25, SignalChem, USA), rabbit anti-GSK3β (1:1000, ab32391, Abcam, USA), or rabbit GSK3β phospho-Y216 (1:150, ab75745 Abcam, USA), and mouse anti-PV (1:5000, 235, Swant, Switzerland). Images of the auditory cortex were captured using confocal microscope (Nikon A1). NIH ImageJ software was used to measure the integrated density of GSK3α, GSK3β and GSK3-phospho (Y279/Y216) in PV-positive interneurons or non-PV neurons. The corrected total cell fluorescence (CTCF) was calculated as previously described [26 (link)]. Results are presented as normalized florescence, which is the ratio of CTCF value of the protein to the CTCF values of corresponding proteins in non-PV neurons of floxed-control mice.