E cadherin apc

Excised mouse trachea was incubated in PBS serum, anti-CD16/32, and indicated antibodies for 4 h at room temperature. The following antibodies were used in different combinations at the indicated dilutions: CD103–Alexa Fluor 594 (Biolegend), 1:50; CD49a-Alexa Fluor 647 (BD Biosciences), 1:25; and E-cadherin–APC (Biolegend) 1:50. Images were acquired on an Olympus FV1000 (Center for Advanced Light Microscopy and Nanoscopy Shared Resource Laboratory) using an Olympus Plan Apo 60×/1.43 NA objective. Fresh, transplant donor–quality human tracheal tissue was obtained from the Human Tissue Core of LungMAP at the University of Rochester. The University of Rochester Institutional Review Board approved and oversees this study (approval no. RSRB00047606). Human trachea was stained for 72 h in PBS serum and the following Biolegend antibodies: hCD103–Alexa Fluor 594, 1:40; hCD8–Alexa Fluor 488, 1:40; hCD49a–Alexa Fluor 647, 1:40; and E-cadherin–APC, 1:40. Human tissue was washed in PBS and fixed in 4% paraformaldehyde for 2 h prior to mounting. Mouse tissue was washed in PBS and mounted unfixed. All whole-mount tissue sections were mounted using Fluoromount-G (SouthernBiotech).

After OCT block equilibration at −20°C, 10 μm slices were obtained using a cryostat, mounted on glass slides, dried for 20 minutes at - 20°C, and fixed in ice-cold acetone at −20°C for 20 minutes. After fixation, slides were dried briefly at room temperature and stored at −80°C until stained or used immediately. For staining, slides were equilibrated at room temperature, washed in 4°C PBS twice for five minutes, blocked in serum-free blocking reagent (Dako) ON at 4°C, followed by staining with CD45.1-AF594 (BioLegend, clone A20, Reference #110756) and E-cadherin-APC (BioLegend, clone DECMA-1, Reference #147312), and CD8a-FITC (BioLegend, clone 53–6.7, Reference #35–0081-U500) diluted in Antibody diluent solution (Dako, S080983-2) overnight at 4°C, stained with DAPI, and mounted with coverslips using Vectashield Vibrance Antifade mounting media (VectorLabs, H-1700). Images were acquired on an Olympus VS200 Slide Scanner (UCSD Microscopy CORE) or on a ZEISS LSM700 confocal microscope. Quantifying P14 CD8 T cell distances for IMAP representation over time was done using a groovy script on QuPath.