Hippocampal Slice Preparation and Analysis

Hippocampal slices were cut (300 µm) with a McIlwain tissue chopper (Mickle, UK) and incubated for at least 1 h at room temperature in oxygenated (95% O₂, 5% CO₂) ACSF as described^{39 (link),40 (link)}. They were kept at 32 °C for another hour before treatment with DHPG (100 µM) for 5 min or with Sal003 (20 µM) for 10 min, before snap-freezing over dry ice. In all instances, similar slices were treated with vehicle as control. Frozen slices were lysed in homogenizing buffer (200 mM HEPES, 50 mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM EDTA, 50 mM NaF, 2 mM Na₃VO₄, 25 mM β-glycerophosphate and EDTA-free complete ULTRA tablets (Roche, Indianapolis, IN)). Western blotting was performed as described by Huang et al.^40.Primary antibodies for western blotting were rabbit anti–p-eIF2α (Ser51) (1: 500, 3398, Cell Signaling and Technology Laboratories, Danvers, MA), mouse anti-total eIF2α (1: 1,000, 2103, Cell Signaling), mouse anti-β-actin (1:10,000, ab8227, Abcam, Cambridge, MA), mouse anti-OPHN1 (1:1,000, sc-374330, Santa Cruz Biotechnology, Santa Cruz, CA) and mouse anti-Arc (1: 200, sc-17839, Santa Cruz Biotechnology). Secondary antibodies for western blotting were peroxidase-conjugated goat anti-rabbit (1:10,000, 111–035–144, Jackson ImmunoResearch) and goat anti-mouse (1:10,000, 115–035–003, Jackson ImmunoResearch).