Ixon em du 897 emccd camera

After 1–2 days in culture, except where noted, wt and GCaMP6f-expressing murine ACC were rinsed with phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde in PBS for 10 min. Cultures were blocked in PBS containing 0.1% Triton X-100 (PBST) and 10% fetal bovine serum (FBS) and incubated overnight at 4°C with antibodies against TH (rabbit, PA5-85167, ThermoFisher, Waltham, MA, USA), phenylethanolamine N-methyltransferase (PNMT) (rabbit, AB110, MilliporeSigma, Burlington, MA, USA), or S100β (rabbit, GA50461-2, Agilent, Santa Clara, CA, USA). For GCaMP6f-expressing cells, the overnight incubation included a green fluorescent protein (GFP) antibody (goat, 600-101-215, Rockland Immunochemicals, Pottstown, PA, USA). Primary antibodies were diluted 1:1000. Cells were then rinsed several times with PBS and incubated for one hour at room temperature in the dark with AlexaFluor 594-conjugated donkey-anti-rabbit and/or AlexaFluor 488-conjugated donkey anti-goat antibodies (ThermoFisher). Secondary antibodies were diluted 1:500 in 10% FBS in PBST that also contained 1 μg/ml bisbenzimide (Hoechst 33342; ThermoFisher) to label nuclei. Cells were rinsed with PBS and brightfield and fluorescence images were acquired using a Nikon TE 2000 epifluorescence microscope equipped with an iXonEM + DU-897 EMCCD camera (Andor).

DNA-PAINT microscopy was performed on a home-built SMLM setup with an Olympus IX81 inverted microscope frame equipped with an Olympus 150× TIRF oil immersion objective (UIS2, 1.49NA). The samples were illuminated in HILO mode^{40 (link)} using a 561 nm laser line (Coherent Sapphire LP) at an illumination density of 0.88 kW/cm² through a 4 L TIRF filter (TRF89902-EM, Chroma Technology) and ET605/70 M nm bandpass filter (Chroma Technology). Signals were detected with an Andor iXon EM+ DU-897 EMCCD camera (Andor, Ireland). SMLM frames were acquired using multi-dimensional acquisition (MDA) mode in Micro-Manager 2.0-gamma^{41 (link)}.

Changes in [Ca²⁺]_i were monitored in cells labeled with the Ca²⁺-sensitive fluorescent indicator, Calcium Green-1(Ex_{480 nm} and Em_{535 nm}) as previously described^{18 (link)}. For dye loading, the cells were incubated with 1 µM Calcium Green-1 for 45 min at 37 °C in a balanced salt solution (BSS) containing 0.1% bovine serum albumin (BSA) and (in mM): 145 NaCl, 5 KCl, 1.2 NaH₂PO₄, 2 CaCl₂, 1.3 MgCl₂, 10 glucose, and 15 HEPES, pH 7.4. For fluorescence monitoring, cells were washed twice with dye-free BSS lacking BSA and placed on the stage of a Nikon TE2000 epifluorescence microscope equipped with a 100X objective. For Ca²⁺ free experiments cells were bathed in Ca²⁺-free BSS Ca²⁺ containing 1 mM EGTA. Fluorescence images of the cells were captured before, during and after application of a stimulus by an iXonEM + DU-897 EMCCD camera (Andor Technology Ltd., Belfast, UK) using the open source microscopy software Micro-Manager (version 1.4, Vale Lab, UCSF, San Francisco, CA). The exposure time of the camera was set to 100 ms and images were captured at a rate of 7.5 Hz. Bright field images were obtained at the start and end of an experiment, and all experiments were carried out at ambient room temperature.