Tracheae Staining and Optical Clearing

Staining and optical clearing of dissected tracheae were performed as described in a previous study (Hirashima and Adachi, 2015 (link)). Briefly, the samples were fixed with 4% PFA in PBS overnight at 4°C. For anti-SOX9 staining, the samples were incubated in 25 mg/mL hyaluronidase (Nacalai Tesque, #18240-36) for 1 h at 37°C, to digest hyaluronic acid. The samples were then blocked in 10% normal goat serum (Abcam, #ab156046) diluted in 0.1% Triton X-100/PBS (PBT) for 3 h at 37°C. The samples were treated with primary antibodies overnight at 4°C, washed in 0.1% PBT, and subsequently incubated in secondary antibodies conjugated to either Alexa Fluor 546 or Alexa Fluor 647 overnight at 4°C. DAPI was used for nuclear counterstaining (Dojindo Molecular Technologies, #D523-10, 1:200 dilution). The samples were mounted with 10 μL of 1% agarose gel onto a glass dish (Greiner Bio-One, #627871) for stable imaging. Then, the samples were immersed in CUBIC-R+ (Tokyo Chemical Industry Co., # T3741) solution for optical clearing. Images were acquired using the confocal laser scanning platform Leica TCS SP8 equipped with the hybrid detector Leica HyD, using a ×40 objective lens (NA = 1.3, WD = 240 μm, HC PL APO CS2, Leica) and the Olympus FluoView FV1000 with a ×30 objective lens (NA = 1.05, WD = 0.8 mm, UPLSAPO30XS, Olympus).