Phosphate buffered saline pbs

For vascular bundle network studies among the whole plant, of the main stem on a 45–50 DAG plants were harvested and fixed in 4% paraformaldehyde (Sigma-Aldrich) in 1 x Phosphate Buffered Saline (PBS, Eurobio), buffer-0,1% tritonX100 (Bioprobe) under vacuum on ice for 2 h. Samples were incubated at 4 °C O/N in the fixative and then washed in PBS and store at 4 °C until use. Thirty μm section were obtain with a HM 650 V Vibratome from MicroMicrotech France. For 3D reconstruction of the node, the 3 cm of the fifth node including upper and lower internode of the main WT stem were fixed as described above and later incubated in 10%, then 20% sucrose for 1 h each, and 30% sucrose overnight at 4 °C. After removing the excess of sucrose, samples were embedded in cryo-embedding medium (NEG-50TM, Thermo scientific) and freeze with liquid nitrogen. Tissue cutting were performed at − 16 °C (CryostarTM NX70, Thermo scientific) and 100 μm section were placed on superfrost slides (Thermo scientific) and stored at − 20 °C or mounted in citifluor AF1 (agar scientific) for confocal imaging.

Alveolar macrophages used for the viral titration were obtained from bronchoalveolar lavage (BAL) of lungs collected from 5 to 7-month-old Large White conventionally bred sows. The animals were bred in accordance with European regulations by the experimental unit of animal physiology of Orfasière (UE PAO), Nouzilly, France. They were serologically tested frequently and known to be free of any common viral infection (swIAV, PRRSV, Porcine Circovirus 2, amongst others). In order to reduce the use of animals, the lungs were collected from pigs slaughtered in the course of the regular management of the experimental unit’s herds. As a consequence, no trial number has been attributed since an experimental authorisation was not requested. Once isolated, the lung airways were infiltrated with 250 mL of phosphate buffered saline (PBS) (Eurobio scientific) supplemented with 2 mM EDTA (Sigma-Aldrich, Saint-Quentin, France). The BAL was then collected, centrifuged, and passed through 40 µm cell strainers. After treatment with erythrocyte lysis buffer (10 mM NaHCO₃, 155 mM NH4Cl, and 10 mM EDTA), AM were washed with PBS, counted and seeded onto sterile plates and flasks for virus titration and propagation. Roswell Park Memorial Institute medium (RPMI) 1640 medium (Eurobio scientific) supplemented with 10% FCS and 2% of SPA solution was used for AM culture.