A1r gaasp inverted confocal microscope

Tissues were fixed in 4% paraformaldehyde, embedded in paraffin wax, and sectioned at 5 µm intervals. Slides were stained with hematoxylin using standard protocols, as previously described (Herriges et al., 2014 (link); Swarr et al., 2019 (link)). Immunofluorescence staining was performed using the following antibodies: anti-NKX2.1 (guinea pig, Seven Hills, 1:500), anti-SOX9 (rabbit, Millipore, 1:100), anti-KI67 (mouse, BD Pharmigen, 1:100), anti-SCGB1A1 (rabbit, Seven Hills, 1:500), anti-TUB1A1 (mouse, Sigma Aldrich, 1:1000), and anti-SOX2 (mouse, Santa Cruz, 1:100). Immunofluorescence for phosphorylated-ATK (pAKT) was performed with anti-pAKT (rabbit, Cell Signaling, 1:100), followed by multi-step detection with the Biotin-Tyramide signal amplification kit (Perkin Elmer) with a 15-min exposure time. TUNEL staining was performed using the TACS-XL Blue Label kit, per manufacturer’s instructions (Trevigen). All slides were mounted with Prolong Gold Antifade medium (Invitrogen), and were then imaged on either a Nikon Eclipse 90i widefield, or Nikon A1R GaAsP Inverted Confocal Microscope. Cell type quanitification based on immunofluorescence microscopy images was performed using the Nikon Elements Advanced Analysis software suite.

The cells were seeded in 8 chamber-slides 24 h before the experiments. Nanoparticles labeled with Cy5 were incubated with the cells at 37 °C and the excess of NPs was removed by washing with PBS. The cells were fixed using 4% PFA (Paraformaldehyde, Electron Microscopy Sciences, Hatfield, PA, USA), and the cell nuclei were counterstained with DAPI (4′,6-diamidino-2-phenylindole, blue). The cells and nanoparticles were prepared, imaged and analyzed using a Nikon A1R GaAsP inverted confocal microscope as described [49 (link),50 (link)].