Tissue processing for histology analysis

Briefly, organs were harvested and fixed in 10% formalin for 4 hr then incubated in 30% sucrose-PBS overnight at 4°C before being embedded in Tissue–Tek OCT compound (Sakura Finetek, Alphen aan den Rijn, Netherlands) and frozen at −80°C. Sectioning was completed on a HM550 Cryostat (Thermo Fisher Scientific, Waltham, MA, USA) at −20°C and 5 μm sections were collected on Superfrost Plus Slides (Thermo Fisher Scientific) and stored at −20°C until use.

Vascular staining using beads was performed on histological sections of lung from MacBlue×Cx3cr1^gfp/+ mice injected 5 min before sacrifice with a mixture of 10 µm Polybead Carboxylate Microspheres and 0.2 µm FluoroSpheres carboxylate. Lung sections were rehydrated with PBS for 10 min, counterstained and mounted with Vectashield Mounting Medium with DAPI (4,6-diamidino-2-phenylindole; Vector Laboratories). Imaging used a Zeiss Axio Microscope (Carl Zeiss, Oberkochen, Germany). CD31 vascular staining was performed on lung cryo-sections from MacBlue×Cx3cr1^gfp/+ mice. A first blocking step was performed with 3% BSA/PBS solution for 30 min. Slides were then incubated for 1 hr at 37°C with the primary antibody rat anti-mouse CD31 (4 µg/ml) (clone MEC 13.3; Becton Dickinson, San Jose, CA, USA) or the isotype control (4 µg/ml) (clone eBR2a; eBioscience). Slides were next incubated with Avidin/Biotin Blocking Kit (SP-2001; Vector Laboratories, Burlingame, CA, USA) and then stained with a biotinylated donkey anti-rat IgG at 3 µg/ml for 30 min at room temperature. After three (5 min) washes in PBS, slides were incubated with Cy3-conjugated streptavidin at 2.6 µg/ml for 30 min at room temperature (Jackson ImmunoResearch Laboratories, West Grove, PA, USA). Slides were counterstained and mounted with Vectashield Mounting Medium with DAPI and analysed by using a Zeiss LSM 710 NLO confocal microscope coupled with 458 nm, 488 nm, and 543 nm lasers to detect ECFP, EGFP, and Cy3 simultaneously on three photomultipliers.

Acquisition settings were identical for both isotype and CD31 staining. Volume rendering was performed using Imaris software (Bitplane, Zurich, Switzerland) and parameters were set according to CD31 isotype staining.