Tc sp8 confocal microscope

The PCLS isolated from CSF1R-eGFP or CSF1R-mApple transgenic birds were imaged floating in DMEM within a 35 mm µ-dish (Ibidi), weighed down with sterile 13 mm coverslips (Scientific Laboratory Supplies Ltd, Coatbridge, UK). Live images were captured using a Zeiss Live Cell Observer with the heated stage set to 37 °C and the imaging chamber set to 37 °C and 5% CO₂. PCLS at 3 days post slice were incubated with either red fluorescent 1 μm latex beads (Thermo Fisher Scientific, Paisley, UK) or 5 × 10⁹ CFU/mL APEC O1-eGFP.
Images of PCLS were also captured using a Zeiss Axio Zoom.v16 in the wells of a 24 well plate at 7X or 16X magnification. Confocal images were captured using a Leica TCS P8 confocal microscope at 200X or 630X magnification and the LAS X software (Leica, Wetzler, Germany) or a Zeiss LSM710 inverted confocal microscope and Zen software (Zeiss).

HEK293 cells with inducible expression of RyR₂ (HEK-tet-RyR₂) were kindly provided by Dr. Wayne Chen. Both C2C12 cells and HEK-tet-RyR₂ cells were cultured in DMEM + 10% FBS at 37°C, 5% CO₂. Transfection of plasmids was conducted using Lipofectamine 3000 regent (Thermo Fisher, L3000008, Dallas, TX, USA) and cells were imaged or fixed for immunostaining 2 days post transfection. For HEK-tet-RyR₂ cells, RyR2 expression was induced by applying tetracycline hydrochloride (0.1 μg/mL) for 18 h. C2C12 cells were fixed with 4% paraformaldehyde and stained with TOM20 rabbit polyclonal antibody (Proteintech 11802-1-AP, Rosemont, IL, USA). The secondary antibody used was anti-rabbit-Alexa Fluor 488 (Thermo Fisher). All imaging experiments were conducted using a Leica TC SP8 confocal microscope. The time-lapse recordings of HEK-tet-RyR₂ cells were captured at 1.5 sec/frame. jRCaMP1b was excited with a 561 nm laser and the emission light was collected at 575–625 nm.