RNAscope in situ hybridization (ISH)

Human postmortem hippocampus (1 control sample of Braak 0 and 3 AD samples of Braak 6) and middle temporal gyrus (2 control samples of each Braak 0, 1, and 2 and 2 AD samples of each Braak 4, 5, and 6) were used for triplex fluorescent ISH. Human RNAscope ISH probes were ordered from Advanced Cell Diagnostics Inc. (ACD, Hayward, CA, USA) for NRXN3 in C2 channel (20 ZZ pairs targeted region 1095–2035 of NM_001105250.2; Cat No. 525431-C2), NLRP3 in C1 channel (30 ZZ pairs targeted region 2627–4008 of {"type":"entrez-nucleotide","attrs":{"text":"NM_004895.4","term_id":"208879434"}}NM_004895.4; Cat No. 478021), and NEUN/RBFOX3 in C3 channel (20 ZZ pairs target region 720–2217 of {"type":"entrez-nucleotide","attrs":{"text":"NM_001082575.2","term_id":"460417306"}}NM_001082575.2; Cat No. 415591-C3). The positive control probes (Cat No. 320868) were POLR2A (C1 channel), PPIB (C2 channel), and UBC (C3 channel). The negative control probe was bacterium (Bacillus subtilis) gene DapB (Cat No. 320871). The cryostat sectioning of postmortem human brain samples, fixation, protease pretreatment, probe hybridization, pre-amplification, amplification, horseradish peroxidase reaction, and fluorescent labeling steps were described previously [39]. Zeiss LSM 880 confocal microscope was used to image fluorescent labeling. Amplification × 20 images (two to three images for each brain sample) were analyzed by FISH v1.0 module included in HALO software with RNAscope ISH setting (Indica Labs, Corrales, NM, USA). The H-score [Σ_bin0–4 (ACD score or bin number × percentage of cells per bin)] were used to calculate mRNA expression for each probe based on the minimum intensity threshold (a value between 0 and 400).