Nel704a001kt

Histopathological analyses were performed on formalin-fixed paraffin-embedded tissue after Mayer’s H&E staining. Immunohistochemistry of tissue sections was performed using the TSA system as recommended by the manufacturer (NEL704A001KT, PerkinElmer). Antibodies for immunohistochemistry were as follows: anti-human SMYD2 (HPA029023; Sigma-Aldrich), anti-murine SMYD2 (21290-1-AP; Proteintech), anti-cleaved caspase-3 (9661; Cell Signaling Technology), and anti-rabbit IgG (7074; Dianova). Nuclei were stained with Hoechst 33342 (H3570; Invitrogen). Imaging was carried out using a Leica laser-scanning confocal microscope or Hamamatsu Nanozoomer 2.0 HT (Hamamatsu).

WT mice were killed through cervical dislocation and their brains removed and frozen by immersion into isopentane cooled to ∼−50 °C on dry ice. Coronal sections were then cut at a thickness of 20 μm on a cryostat at the level corresponding to NAc (NAc; AP: +1.4 to +0.6 mm), thaw mounted on glass slides and stored at −20 °C until used for immunofluorescent staining. Immunofluorescence was carried out on the fresh–frozen, 20-μm thaw-mounted sections using the Tyramide Signal Amplification (TSA) Plus system (Perkin Elmer, NEL704A001KT). In brief, all slides were fixed using ice-cold 4% paraformaldehyde pH 7.4 (10 min), permeablized in 0.01% Triton X-100 in 0.1 M PBS (5 min) and incubated in 2% hydrogen peroxide diluted in 0.1 M PBS (vol/vol) (15 min). Subsequently, all slides were then blocked (1 h) using the TSA blocking buffer and incubated overnight (4 °C) in rabbit antibody to BDNF (1:500, Santa Cruz Biotechnology, sc-546), washed (3 × 5 min in 0.1 M PBS with 0.05% Tween-20, vol/vol) and incubated for 1 h at 21–23 °C with horseradish peroxidase-conjugated goat antibody to rabbit IgG (1:500, 711-036-152, Jackson ImmunoResearch). Slides were again washed and the TSA reaction was performed to detect BDNF as described in the kit (TSA-Alexa Fluor 488 1:50). Slides were coverslipped in ProLong Gold antifade reagent with DAPI (P-36931, Invitrogen).

RNAscope, high-resolution RNA in situ hybridization (97 (link)), was performed on FFPE sections using the RNAscope Multiplex Fluorescent V2 Assay (323100, ACDBio). Slides were boiled in Target Retrieval Reagents for 15 minutes and treated with Protease IV for 30 minutes. Probes were specific for Mm-Cbfa2t3 (43601-C2, ACDBio), Mm-Muc2 (315451, ACDBio), and Mm-Neurog3 (422401, ACDBio); 1:750 TSA Cy3 (NEL704A001KT, PerkinElmer) and TSA Cy5 (NEL75A001KT, PerkinElmer) were used for probe visualization.