Rabbit anti pro sftpc

After preincubating for 15 minutes with anti-mouse CD16/32 antibodies (eBioscience), Fixable Viability Stain 780 (BD Bioscience) was used to exclude dead cells. Subsequently, lung cells were labeled with antibodies. For staining indirectly labeled antibodies, the primary antibodies were stained for 30 minutes and washed out followed by the secondary antibodies staining. The antibodies used were as follows: APC/Fire 750 anti-mouse F4/80, BV650 anti-mouse/human CD11b, BV421 anti-mouse Ly6G, PE/Cyanine7 anti-mouse Ly6C, BV421 anti-mouse Ki67, PE anti-mouse PDPN, APC anti-mouse CD31, APC anti-mouse CD45, FITC anti-mouse CD326/EPCAM, BV510 donkey anti-rabbit IgG, AF488 donkey anti-rabbit IgG, rabbit anti-pro-Sftpc (Millipore), rabbit anti-mouse CCSP (Proteintech), CF633-α-Bungarotoxin (Biotium) (all others from Biolegend). Debris and aggregates were≠ excluded and live cells were detected on a LSRFortessa (BD Biosciences) and then analyzed by FlowJo X 10.0.7 software (Tree Star Inc.). BD FACS Aria II appliances were used for AT2 lineage–labeled cell sorting; the sorting strategies are shown in the figures. For isolation of lung mesenchymal cells, cells negatively isolated by a CD31 via MagCellect Magnet column (R&D Systems) were further negatively sorted by EPCAM and CD45 via a MagCellect Magnet column.

PLISH barcoding was performed as described above. Subsequently, the sample was washed 3 × 5 min with PBST at RT, and incubated in blocking solution (50 μl/ml [5%] normal goat serum, 1 μl/ml [0.1%] Triton X-100, 5 mM EDTA and 0.03 g/ml [3%] BSA in PBS) at RT for 1 hr. The sample was then incubated with primary antibody (Rabbit anti-pro-Sftpc, Millipore, 1:500 or Rabbit anti-Cytokeratin 5, Abcam Ab193895, 1:400) in blocking solution at 37°C for 2 hr under gentle rocking, washed 4 × 5 min with PBST at RT, and incubated with secondary antibody (Goat anti-Rabbit-Cy5, Jackson Lab, 1:250) and DAPI (1:1000) in blocking solution at RT for 1 hr. The sample was washed 3 × 5 min in PBST at RT and mounted in H-1000 Vectashield.