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).
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