The effect of prolonged ethanol exposure on constitutive recycling of 5-HT1ARs was determined as described previously (Luessen et al. 2016 (link)). Surface 5-HT1ARs were labeled with rabbit anti-HA (Novus Biologicals cat#NB600–363) for 60 min at 4°C, followed by warming up cells to 37°C to induce receptor internalization. Any remaining, non-internalized antibody-bound 5-HT1ARs on the surface were blocked at 4°C using non-fluorescent secondary antibody goat anti-rabbit IgG-HRP (Cell Signaling Technology cat#7074P2). Cells were again warmed up to 37°C to allow internalized 5-HT1ARs to recycle back to the surface in 5, 15, or 30 min. The recycled 5-HT1ARs on the surface were probed with goat anti-rabbit Alexa Fluor 488 (Invitrogen cat#A32731TR). After fixation and permeabilization, internal (non-recycled) 5-HT1ARs were labeled using goat anti-rabbit Alexa Fluor 594 (Invitrogen cat#A-11012). Similar to internalization, the amount of recycling was calculated as a ratio of the intensity of Alexa Fluor 488 over that of Alexa Fluor 488 and 594 combined (recycled 5-HT1ARs/total surface 5-HT1ARs before assay) at an indicated time. Data are presented relative to basal 5-HT1AR levels for each treatment condition.