Axioobserver z1 stand

Acss2 ED and WT littermate MEF were plated onto chamber slides, incubated for 24 hr, fixed with ice cold methanol for 15 min at -20°C, then rinsed with 1x PBS. Cells were permeabilized with 0.5% Triton X-100 in 1x PBS for 10 min at room temperature, then rinsed once with 1x PBS. Slides were blocked for 30 min at room temperature in 5% normal goat serum, 1% fish skin gelatin (Cat. # G7765, Sigma, St. Louis, MO), 1% BSA (Cat. # A3059, Sigma, St. Louis, MO) in 1x PBS, then incubated in anti-Acss2 antibody (Cat. # 3658, Cell Signaling Technology, Danvers, MA) diluted 1:400 in blocking solution for 2 hr at room temperature. Slides were washed 3 times in 1x PBS, once in 1x PBS + 0.1% Triton X-100 for 1 min each wash, followed by a 1 hr room temperature incubation with Alexa 555 goat anti-rabbit secondary antibody (1:400 dilution; Cat. # 4413, Cell Signaling Technology, Danvers, MA). Slides were washed 3 times for 1 min in 1x PBS, then mounted with Vectashield containing DAPI (Cat. # H1200NB, Vector Labs, Burlingame, CA). Fluorescent images were obtained with 20X magnification using a Zeiss LSM 880 on an AxioObserver Z1 stand with Zen 2.3 Pro software (University of Texas Southwestern O’Brien Kidney Center Core).

SNAP‐β₂AR cells were seeded onto poly‐D‐lysine‐coated (10 µg/ml) 8‐well Nunc™ Lab‐Tek™ chambered coverglass (No. 1.0 borosilicate glass bottom) in DMEM supplemented with 10% FBS at a density of 10–15,000 cells per well 2 days prior to experiment. On the day of the experiment, media were replaced with labeling media which contained SNAP‐Surface® Alexa Fluor® 488 or 647 (New England Biolabs) at a final concentration of 0.5 μM (for 30 min at 37ºC). Cells were then washed in warm HBSS before a final addition of 200 μl of HBSS per well.
Cells were imaged on a Zeiss LSM880 with a Zeiss Axio Observer Z1 stand (Carl Zeiss) with a 40× C‐apochromat NA1.2 water immersion objective. Excitation was via 488 nm Argon and 633 nm helium‐neon laser lines with a 488/561/633 multibeam splitter and emission collected using a 493–628 bandpass or 638–737 bandpass. The pinhole was set at 1 Airy unit for the longer wavelength and laser power and gain and offset settings kept constant within experiment to allow comparison. Cells were imaged live at 24ºC following a 30 min pre‐incubation at 37ºC in the presence of fluorescent ligand (100 nM) following a 30 min pre‐incubation at 37ºC in the presence or absence of 10 μM propranolol. Equatorial plane images were made and four images captured per condition per experiment using ZEN 2012 software (Carl Zeiss).