Chunk #44 — MATERIALS AND METHODS — Immunohistochemistry

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Polygenic risk for alcohol use disorder affects cellular responses to ethanol exposure in a human microglial cell model.

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For immunocytochemistry analysis, the cultured cells were fixed using freshly prepared buffered 4% paraformaldehyde. Subsequently, after permeabilization for 10 min with 0.2% Triton X-100 in phosphate-buffered saline (PBS) and blocking with goat serum, the cell cultures underwent an antibody-labeling procedure. Specifically, the cultures were incubated overnight at 4°C with the following primary antibodies, which were diluted in blocking buffer: rabbit anti-CD235a (Thermo Fisher Scientific, catalog #PA5-27154, RRID: AB_2544630), mouse anti-CD43 (Thermo Fisher Scientific, catalog #14-0439-82, RRID: AB_763493), chicken anti-IBA1 (Aves Labs, catalog #IBA1-0020, RRID: AB_2910556), mouse anti-TMEM119 (Cell Signaling Technology, catalog #41134S, RRID: AB_3094467), rabbit anti-P2RY12 (Sigma-Aldrich, catalog #HPA014518, RRID: AB_2669027), rabbit anti-PU.1 (Cell Signaling Technology, catalog #2266S, RRID: AB_10692379), rabbit anti-CX3CR1 (Bio-Rad, catalog #AHP1589, RRID: AB_2087421), mouse anti-CD68 (Thermo Fisher Scientific, catalog #MA5-13324, RRID: AB_10987212), mouse anti-CLEC7A (R&D Systems, catalog #MAB1859, RRID: AB_2081791), and mouse anti-γH2AX (phospho-histone Ser139, Millipore Sigma, catalog #05-636, RRID: AB_309864), chicken anti-MAP2 (EMD Millipore, catalog #AB5543, RRID: AB_571049), and mouse anti–synapsin 1 (Synaptic Systems, catalog #106011, RRID: AB_2619772). After incubation with primary antibodies and subsequent PBS washes, the cultures were exposed to secondary antibodies labeled