Dsu spinning disk confocal

We grew cells to ~70% confluency on 12 mm glass coverslips placed in 60 mm plates over three days and then starved with serum-free media overnight. The cells were then incubated with 500 nM imaging agent for 6 hours at 37 °C in serum-free media at indicated zinc concentration, washed thrice with PBS, fixed in 4% paraformaldehyde in PBS, permeabilized with 0.1% Triton X-100 in PBS, and mounted in Prolong Gold with DAPI (Invitrogen). For immunofluorescence studies, cells were permeabilized with 0.1% Triton X-100 in PBS, followed by incubation with 1:250 dilution of anti-hPRLR antibody (sc-20992) overnight, and briefly with a 1:1000 dilution of Alexa 488 labeled mouse anti-rabbit antibody (Invitrogen) before mounting.
Frozen sections of human tissue specimen for hPL*-Cy5 binding were probed with 500 nM hPL*-Cy5 in the presence of 20 μm zinc overnight, washed thoroughly with dPBS, and mounted using the immnunofluorescence protocol. Data was analyzed with an Olympus FV1000 (Olympus America). After processing of frozen xenograft mouse tumors by the HTRC, we used mounted tissue protocol described above for confocal analysis. These samples were analyzed using a DSU Spinning Disk Confocal (Olympus America). All data were processed using ImageJ software (v1.42q, NIH).