Psd95

The paraffin‐embedded sections were rehydrated using graded alcohols. After antigen retrieval buffer and autofluorescence eliminator reagent (Beyotime Biotechnology, Shanghai, China) were added, MeCP2 antibody (dilution: 1:50) (Santa Cruz Biotechnology, Texas, USA), PSD95 (dilution: 1:100) (ABclonal, Wuhan, China), and secondary antibody included either goat anti‐rabbit Alexa Fluor 594 (ABclonal, Wuhan, China) were utilized. 4’,6‐diamidino‐2‐phenylindole (DAPI) (Beyotime Biotechnology, Shanghai, China) was used for nuclear staining. As for MeCP2, average fluorescence intensity was analyzed and immunopositive cells in each field were calculated using Image‐Pro Plus software (Media Cybernetics, Inc., USA). With respect to PSD95, the density of PSD95 puncta was analyzed using Image‐Pro Plus (Media Cybernetics, Inc., USA) and ImageJ software (Universal Imaging Corporation, USA) by an observer blinded to the experiments.

After extraction and separation by SDS‐polyacrylamide gel electrophoresis in 10% Tris‐glycine gels, the protein samples were transferred to a nitrocellulose membrane. Primary antibodies including MeCP2 (Santa Cruz Biotechnology, Texas, USA) (dilution: 1:500), PSD95 (1:500) (ABclonal, Wuhan, China), CREB1 (Cell Signaling Technology, Inc., Danvers, USA) (dilution: 1:700), p‐CREB1 (ser133) (Cell Signaling Technology, Inc., Danvers, USA) (dilution: 1:700), and GAPDH (Proteintech Group, Inc, Wuhan China) (dilution: 1:10000) were incubated at 4℃ overnight. Subsequently, IRDye purified secondary antibody (dilution: 1:10000) was incubated. Immunopositive bands were visualized at Ex/Em=778nm/795nm.