Vectashield containing dapi h 1200

For immunofluorescence staining, cells were grown on coverslips and cultured in the supplemented media at 37°C in an atmosphere of 5% CO₂. Cells were fixed with ice-cold methanol for 10 min, after which excess methanol was removed. After three subsequent washes with PBS, cells were blocked and permeabilized for 1 hour with PBS containing 1% BSA, 5% goat serum and 0.05% Triton X-100 (Sigma–Aldrich, St. Louis, Missouri, USA). The cell coverslips were incubated with primary antibody (mouse anti-GHR B10 (Santa Cruz, CA, USA), mouse anti-PrlR, clone 1A2B1 (Invitrogen, Carlsbad, CA, USA) and rabbit anti-PrlR M-170 (Santa Cruz, CA, USA) overnight at 4°C, washed again 3 times with PBS, and incubated for 1 h at room temperature in the dark with the secondary antibodies alexa fluor 488 goat anti-mouse and alexa flour 594 goat anti-rabbit (both from Invitrogen, Carlsbad, CA, USA). Finally, the slides were washed, mounted with Vectashield containing DAPI (H-1200, Vector Laboratories, CA, USA) and stored at 4°C in the dark. Controls for specificity and background staining were performed by incubating with mouse IgG or rabbit IgG antibodies. Image acquisition was performed by confocal laser scanning microscopy (CLSM).