Dm4000b microscope

Sections from formalin-fixed and paraffin-embedded xenografted tumors were stained with hematoxylin and eosin. Immunohistochemical analysis was performed using antibodies directed against LC3b (Nanotools (Teningen, Allemagne) #0231–100; mouse mAb; clone 5F10; 1/100) and cleaved caspase 3 (R&D Systems (Minneapolis, MN, USA) #AF835; polyclonal rabbit Ab; 1/500). Karpas-299 cells, treated or not with 500 nM crizotinib for 24 h, were included in low melting agarose and then formalin-fixed and paraffin-embedded. Sections were immunostained with antibodies directed against LC3b (Nanotools) and nuclei were counterstained with hematoxylin. Antibody binding was detected with the streptavidin-biotin-peroxidase complex method (Vector Laboratories (Burlingame, CA, USA)). Pictures were taken using either a Leica DM4000B microscope (Wetzlar, Germany) or a Pannoramic 250 device (3DHISTECH) (Budapest, Hungary).

Placental explants were fixed in formalin during 24 h at room temperature and embedded in paraffin. Tissue sections (5 μm) were de-waxed using xylene and alcohol and epitope retrieval was carried out using citrate buffer (pH 6) at 95°C during 20 min. Sections were re-hydrated using TBS 0.01% Tween 20 for 5 min and blocked with 2.5% horse serum for 20 min. Immunostainings were performed with the following antibodies: rabbit anti-Cytokeratin-7 (Genetex; 2 μg/mL), mouse anti-Vimentin (Santa-Cruz; 2 μg/mL) and mouse anti-placental alkaline phosphatase (Biolegend; 1 μg/mL). Immunostaining for hCMV was performed as previously described (Benard et al., 2014 (link)), using a mouse monoclonal antibody directed against the hCMV IE antigen (clone CH160, Abcam). Secondary antibody-coupled to biotin was then used prior to Vectastain RTU elite ABC Reagent (Vector laboratories) and staining by diaminobenzidine (DAB). Sections were finally counter-stained with hematoxylin. Image acquisition was performed on a Leica DM4000B microscope or on a Panoramic 250 scanner (3DHISTECH).