Alexa fluor594 conjugated secondary antibodies

Sections were blocked with 2.5% normal horse serum and incubated with primary antibodies (anti-α7nAChR and anti-AQP1 from Santa Cruz Biotechnology; anti-calbindin from Abcam) overnight at 4 °C. After washing, the sections were incubated with Alexa Fluor488-conjugated and/or Alexa Fluor594-conjugated secondary antibodies (Vector Laboratories). Fluorescence was visualized using a Fluoview 1000 (IX-81) confocal microscope (Olympus), and the images were quantified by using ImageJ (NIH).

Matrigel-embedded hiAT2 organoids were collected and centrifuged (3000 rpm for 3 min) and then treated with VitroGel Cell Recovery Solution (TheWell Bioscience, NJ, USA MS03-100) for 15 min at 37°C to dissociate the cells from the Matrigel. The dissociated cells were fixed with 4% paraformaldehyde (20 min at room temperature), blocked using 10% donkey serum and then incubated overnight with primary antibody at 4°C. The primary antibodies used are listed in Supplementary Table 2. After washing, the cells were incubated with anti-mouse Alexa Fluor 488-conjugated or anti-rabbit Alexa Fluor 594-conjugated secondary antibodies (Vector Laboratories, CA, USA). Finally, the nuclei were stained with Hoechst (Invitrogen, 33342) and visualized using a Zeiss LSM880-Airyscan Elyra confocal microscope or a confocal laser-scanning microscope (Olympus Corp., Tokyo, Japan; FV3000).