Anti fluorescence quenching sealing tablets

The airway organoids were harvested and fixed in 4% paraformaldehyde at 4 °C overnight. For the section IF, the fixed organoids were dehydrated, paraffin-embedded and sectioned. For the whole-mount IF, the fixed organoids were permeabilized using 0.5% Triton X-100 and blocked with 5% bovine serum albumin (BSA) for 1 h at room temperature (RT). The organoids were then incubated with primary antibodies, including ace-tubulin (Santa Cruz, Santa Cruz, CA, USA, sc-23950), Krt5 (Abcam, CB, Waltham, MA, USA, ab52635), and DNAH5 (Abcam, ab234826), at 4 °C overnight. The next day, the organoids were washed three times and incubated with Hoechst 33342 (Invitrogen, Life Technologies, Carlsbad, CA, USA, H3570) and the secondary antibodies labelled with Alexa Fluor 488 (Invitrogen, A11001) or Alexa Fluor 594 (Invitrogen, A11012) for 1 h at RT. Finally, the organoids were washed three times and sealed with anti-fluorescence quenching sealing tablets (YEASEN, Shanghai, China, CA, 36307ES08). The images were acquired using a laser-scanning confocal microscope (Olympus, Waltham, MA, USA). The quantification of the expression intensities of the different markers was performed using ImageJ software.

After perfusion with 4% paraformaldehyde, the brains were removed and fixed with 4% paraformaldehyde overnight. Next, they were dehydrated in an ethanol gradient (50%, 70%, 80%, 90%, 95%, 100%) and xylene. The tissues were embedded in paraffin wax and sliced into 4-μm sections that were mounted on slides. The slides were deparaffinized and rehydrated in xylene and an ethanol gradient. Antigens were retrieved via thermal repair in a citrate buffer. Then, the slides were blocked at room temperature for 30 min and incubated with the primary antibody of GAP43 (1:500, Abcam) or SNAP25 (1:500, Abcam) overnight at 4 °C. After washing, the slides were incubated with secondary anti-rabbit IgG H&L (Cy3) antibody (preabsorbed and used at 1:500, Abcam) for 1 h at room temperature. Finally, the slides were counterstained with DAPI (Beyotime, Shanghai, China) and sealed with anti-fluorescence quenching sealing tablets (Yeasen, Shanghai, China). Images were obtained using a confocal microscope (Leica TCS SP5 II).