Triton x 100

At P15, wt and Mecp2^−/y mice were perfused intracardially with antigenfix (Diapath, Italy) and brains were sliced (70 µm thickness) using a Leica VT1000S vibratome (Leica Microsystems, Germany). Selected sections from wt and Mecp2^−/y hippocampi were processed in parallel for immunohistochemistry under identical conditions. Sections were incubated for 1 h at room temperature, with a mixture of 5% normal goat serum (NGS, Jackson ImmunoResearch Laboratories, Inc., PA, USA) in 0.1 M phosphate buffered saline (PBS, Life Technologies, CA, USA) with 0.3% Triton X-100 (Sigma-Aldrich, MO, USA). They were then incubated overnight at 4 °C with the anti-KCC2 antibody (rabbit; 1/800; US Biological, MA, USA) diluted in PBS with NGS 1% and 0.1% Triton X-100. After being washed in PBS, sections were incubated for 1 h at room temperature with the fluorescent-labeled secondary antibody Alexa Fluor 555 (1/1000, Life Technologies, CA, USA) diluted in PBS. Sections were finally mounted on slides and imaged on a SP5X Leica confocal microscope (Leica Microsystems, Germany) using identical settiNGS (2 hippocampal hemispheres of 3 sections per animal). KCC2 immunofluorescence analysis of the whole CA3 pyramidal layer within the image was performed blindly using the open-source platform Fiji (1.50e, Java 1.8.0_60, 64-bit; https://fiji.sc/)^{103 (link)}.

Ferric chloride hexahydrate (FeCl₃.6H₂O) was purchased from Fisher Chemicals and tannic acid (C₇₆H₅₂O₄₆) was from Loba Chemie; Dulbecco’s modified Eagle’s medium (DMEM), Dulbecco’s modified Eagle’s medium Ham’s F-12 (DMEM/F-12), and Trypsin-EDTA were purchased from Caisson Laboratories. Fetal bovine serum (FBS) was from HyClone^TM Thermo Fisher Scientific. Collagen type I, ribonuclease A (Rnase A), triton X-100, propidium iodide (PI), and annexin V (FITC) apoptosis detection BioAssay™ kit were purchased from US Biological. Sodium azide (NaN₃) and potassium thiocyanate (KSCN) were procured from BDH Laboratory. Dimethyl sulfoxide (DMSO) and monodensylcadavarine (MDC) were purchased from Sigma-Aldrich; nitric acid (HNO₃) and ethanol were procured from ACL Labscan; and formalin was purchased from Gammaco. 3-dimethylthiazol-2,5-diphenyltetrazolium bromide (MTT) and acrylamide/bis-acrylaminde were purchased from Bio Basic Inc. and etramethylethylenediamine (TEMED) was procured from Invitrogen. Hydrochloric acid (HCl) was purchased from J.T. Baker; methanol was procured from Northern Chemicals and Glasswares Ltd.; and acridine orange was purchased from AMRESCO. LC3B polyclonal antibody and goat anti-rabbit lgG (H + L) secondary antibody, DyLight 488, were purchased from Thermo Fisher Scientific.