Antibody against iba 1

LPS, ATP, BzATP (catalog number B6396), and the antibody anti-α-tubulin (catalog number T6199) were purchased from Sigma-Aldrich (Madrid, Spain). Anti-NeuN antibody (catalog number MAB377) were obtained from Merck Millipore (Tullagreen, Ireland). A438079 (catalog number 2972/10) was obtained from Tocris BioScience (Bristol, United Kingdom). Monoclonal anti Aβ 4–10 residues clone WO2 (catalog number MABN10) was purchased from Merck Chemicals (Madrid, Spain). The commercial antibody for P2X7R (catalog number APR-004) was purchased from Alomone Labs (Jerusalem, Israel). Antibodies against enhanced green fluorescent protein (EGFP) were obtained from Thermo Fisher (catalog number A11122) and AVESLab (catalog number GFP-1020) (Tigard, OR, United States). Red Fluorescent 2 μm diameter microspheres (catalog number F8826) (Life technologies). Antibody against Iba-1 (catalog number 019-19741) was provided from Wako (Richmond, VA, United States). CytoD (catalog number sc-201442) was provided by Santa Cruz (Dallas, TX, United States). Selective P2X7R antagonist GSK 1482160A was provided by GlaxoSmithKline (Madrid, Spain)

Brain slices of the VTA were obtained from opioid-naïve and -dependent mice with or without minocycline treatment and prepared for immunocytochemical detection (Supplementary Material and Methods). For microglial staining, sections were incubated overnight with an antibody against IBA-1 (1:2000; Wako, Richmond, VA) at 4 °C followed by a goat anti-rabbit secondary antibody conjugated to Alexa Fluor 488 (1:1000, Millipore). For KCC2 staining, sections were incubated overnight with a rabbit antibody against KCC2 (1:500; Millipore) at 4 °C followed by a highly cross-adsorbed donkey anti-rabbit IgG conjugated to Alexa Fluor 594 (1:500; Invitrogen, Grand Island, NY). For KCC2 quantification, fluorescence intensity (total intensity per region of interest) was measured with Image J Software. For IBA1 quantification, the degree of microglial activation in the VTA was measured using a semi-quantitative method, based on defined morphological criteria, including cell body size, number of processes, and increasingly ramified morphology (Brettschneider et al, 2012 (link)). The level of microglial activation was scored on a linear scale (0–4) ranging from resting microglia (0) to highly activated (4).