Goat biotinylated anti pdgfr β

Vibratome lung sections were blocked with 5% normal goat serum in 0.5% Triton X-100/PBS (PBS-T) at 4°C overnight. Sections were then incubated in primary antibodies for 1–3 days at 4°C, washed in PBS-T, incubated in secondary antibodies overnight at 4°C, washed again in PBS-T, and placed on slides in mounting media (Dako). Primary antibodies used were rat anti-MECA-32 (1:15, Developmental Studies Hybridoma Bank [DSHB]), rabbit anti-GFP (1:250, Invitrogen), rabbit anti-SMMHC (1:250, Biomedical Technologies), rabbit anti-PDGF-B (1:100, Abcam), goat anti-KLF4 (1:100, R&D Systems), rabbit anti-HIF1-α (1:100, Novus Biologicals), rabbit anti-pH3 (1:200, Millipore), rat anti-CD68 (1:200, Bio-Rad), directly conjugated Cy3 or fluorescein isothiocyanate (FITC) mouse anti-SMA clone 1A4 (1:250, Sigma), and goat biotinylated anti-PDGFR-β (1:10; R&D Systems). ABC Elite reagents (Vector Laboratories) and fluorescein tyramide system (PerkinElmer) were used to amplify the biotinylated PDGFR-β staining as described previously (Greif et al., 2012 (link); Metzger et al., 2008 (link)). Secondary antibodies were conjugated to Alexa 488, Alexa 564, or Alexa 647 (Invitrogen) or to DyLight 649 (Jackson Laboratory) fluorophores (1:500). Nuclei were stained with DAPI (1:500).