Alexa 488 and alexa 568 labeled secondary antibodies

Airway organoids were fixed with 4% paraformaldehyde (PFA) and then paraffin-embedded after routine dehydration. Sections (5 µm) of the airway organoids were stained with hematoxylin and eosin (H&E) and measured for their immunofluorescence (IF). For IF staining, sections that had been deparaffined and rehydrated were boiled in 10 nM sodium citrate buffer (pH 6.0) at 95 °C for 10 min for antigen retrieval. After rinsing in PBS, sections were blocked in 5% goat serum in PBS (blocking solution) for 1 h at room temperature. Primary antibodies diluted in blocking solution were incubated overnight at 4 °C. Alexa 488- and Alexa 568-labeled secondary antibodies (Invitrogen) were incubated in a dark box at 1:500 dilution in blocking solution for 1 h at room temperature. The slides were mounted in 70% glycerol with DAPI diluted at 1:1500 for simultaneous antifade and nucleus staining. For staining of isolated airway organoids, the cells were spun on slides, fixed with 4% PFA for 10 min, permeabilized with 0.1% Triton X-100 in PBS for 15 min, and then blocked with blocking solution for 1 h at room temperature. Staining for VD receptor (VDR) was performed as described above.

Phenylephrine (SIGMA), MG132 (SIGMA). Sarcomeric Alpha Actinin (EA-53, Abcam), Cardiac Troponin I (Abcam), Gapdh (clone 6C5, Millipore), Myc (D84C12, CST), RNA polymerase II (8WG16, Millipore), Btg2 (custom-made polyclonal antibody by immunizing rabbits, Cyclex), Cnot7 (18W, Santa-Cruz), α-Tubulin (MBL), anti-FLAG (M2, SIGMA), and Alexa 488-and Alexa 568-labeled secondary antibodies (Invitrogen).