Mab360

Proteins of primary cultured neurons and homogenized hippocampal tissues were extracted in RIPA buffer (ThermoFisher) supplemented with a protease and phosphatase inhibitor cocktail (ThermoFisher). After centrifugation (4 °C, 13,000 rpm, 15 min), the supernatant was collected into a new 1.5 mL tube and stored at − 80 °C. Total proteins (15–30 μg) were resolved on 10–15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and electrotransferred with transfer buffer to polyvinylidene fluoride transfer membrane (Bio-Rad). Then, the membranes were blocked in TBST (10 mM Tris- HCl, pH 7.4, 150 mM NaCl, 0.1% Tween 20) containing 5% BSA and incubated with the appropriate primary and secondary antibodies. PSD-95, MAP2, GFAP, Iba1 and Nlgn3 were detected in the membranes using anti-PSD95 (Abcam, ab18258), anti-MAP2 (Sigma-Aldrich, M9942), anti-GFAP (Millipore, MAB360), anti-Iba1 (Invitrogen, PA5-27436), and anti-Nlgn3 (Santa Cruz, SC-271880), respectively. β-Actin antibody (Sigma-Aldrich, A1978) was used as the standard. Protein bands were detected using the Clarity™ Western ECL Substrate (Bio-Rad). Bands were captured using Image Lab 6.0.1 (Bio-Rad). Densitometry analyses are presented as the ratio of protein to β-actin protein, which was compared with the controls and normalized.

Free floating sections were first washed in 1 × PBS for 10 min before being transferred to blocking solution (10% donkey serum, 0.3% Triton X-100 in 1 × TBS) for 1 h. The sections were incubated in primary antibody diluted in incubation solution (5% donkey serum, 0.3% Triton X-100 in 1 × TBS) overnight at 4 °C. Sections were then washed three times in 1 × TBS before being transferred to the proper secondary antibody diluted in incubation solution for one hour at room temperature. Then, sections were again washed three times in 1 × TBS before being mounted to slides. Slides were then cover-slipped using VECTASHIELD Antifade Mounting Medium with DAPI (Vector Labs, H-1200-10) and imaged with an Olympus IX-83 Confocal Laser Scanning Microscope (Olympus Life Sciences, Tokyo, Japan). Antibodies and their dilutions were as follows: mouse anti-GFAP (clone GA5, Millipore, MAB360, 1:100); rat anti-CD44 (clone IM7, Invitrogen, 14-0441-82, 1:200); donkey anti-mouse Alexafluor 488 (Invitrogen, A21202, 1:1000); and donkey anti-rat Alexafluor 488 (Invitrogen, A21208, 1:1000).