Donkey anti rabbit igg conjugated to alexa fluor 594

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).

Slides with cryopreserved brain sections were rehydrated in PBS and underwent blocking in 0.25% Triton X-100 and 5% BSA in PBS for 1 h at room temperature. Following blocking, the slides were incubated at 4 °C for 24 h with the following primary antibodies: (I) rabbit polyclonal anti-ERα antibody (1:2000; MilliporeSigma, 06-935) (II) mouse monoclonal anti-NeuN conjugated to AlexaFluor-488 (1:500; MilliporeSigma, MAB377X). Slides were subsequently incubated in the dark for 2 h at room temperature with secondary antibody donkey anti-rabbit IgG conjugated to AlexaFluor-594 (1:250; Invitrogen, A-21207). After washing with PBS, the slides were counterstained with DAPI (1:1000) and mounted with Mowiol 4-88 mounting medium (MilliporeSigma). Imaging was performed using a Leica TCS SP8 confocal microscopy system (Leica Microsystems CMS GmbH).

Primary VS cells were seeded at 10,000 cells/well in SCM on 16-well culture slides precoated with 0.01% PLO and laminin at 5% CO₂ and 37°C. After 24 h, cells were treated with CUDC907 (100 nM) or 0.0005% DMSO (vehicle) in D10 media. Cells were fixed, permeabilized and blocked for 2 h at RT. Slides were exposed to primary antibodies overnight at 4°C, secondary antibodies at RT for 2 h, and DAPI nuclear stain (4′,6-diamidino-2-phenylindole; ab104139, Abcam) for 15 minutes. Slides were cover-slipped with anti-fade mounting medium (Sigma). Primary antibodies used were rabbit anti-YAP (1:200; Cell Signaling, #14074) and rabbit anti-p21 (1:100; ThermoFisher, MA5-14949) and donkey anti-rabbit IgG conjugated to AlexaFluor 594 (1:200; ThermoFisher). Images were obtained with the Leica SP5 Inverted Confocal Microscope (40X oil immersion lens) and assessed qualitatively.