Alexa 633 secondary antibody

Transgene expression was confirmed by visual inspection of tissue from paraformaldehyde-perfused mice. Following the completion of Ca²⁺ imaging sessions, mice were anesthetized by intraperitoneal injection of ketamine/xylazine (100 mg/kg and 10 mg/kg, respectively) and the chest cavity opened, exposing the heart. The heart was accessed by a needle and the animal was perfused at 2 ml/min with a 0.1 M phosphate-buffered (PB) solution followed by paraformaldehyde (4% by volume in PB) until the perfusate exiting an opening from the pulmonary artery ran clear. The cerebellum was removed by dissection and sliced into 80 µm sections in cold PB. When necessary, HA immunostaining was used to confirm hM4d expression. Samples were first incubated with anti-HA antibody (#ab9110, Abcam, Cambridge, UK), followed by an Alexa 633 secondary antibody (Thermo Fisher Scientific, Waltham, MA). DAPI (D1306, Thermo Fisher Scientific) counterstaining was used to identify cell locations in some cases. Images were collected on a confocal microscope (LSM 780 Axio Imager 2; Zeiss, Oberkochen, Germany) using 488 nm excitation and 493–598 nm emission for GCaMP6f, 633 nm excitation and 638–747 nm emission for Alexa 633, 405 nm excitation and 410–507 nm emission for DAPI, and 514 nm excitation and 519–620 nm emission for YFP.

For PM staining, cells were blocked with 1% BSA-PBS and incubated with HA.11 antibody (#901515, Biolegend, San Diego, CA, USA, 1:1000) at 4 °C on living cells. Next, cells were fixed (4% PFA) followed by Alexa-488 secondary antibody (#A-11001, Thermo Fischer Scientific, 1:500). Next, cells were fixed again, followed by permeabilization and HA.11 primary antibody (1:1000). Total anti-HA stained CFTR was visualized using Alexa-633 secondary antibody (#A-21050, Thermo Fischer Scientific, 1:500). Nuclei were stained with DAPI (4′,6-diamidino-2-fenylindole, #D1306, Thermo Fischer Scientific, 1:2000) and sections analyzed by confocal microscopy.