Ix70 inverted fluorescence microscope

Tissue samples were fixed in 4% PFA at RT for 1 h, washed in PBS, dehydrated in 30% sucrose overnight, O.C.T.-embedded (Sakura Finetek) and frozen with ice-cold isopentane (Sigma Aldrich) then stored at −80°C. 7–10 μm thick sections were cut (Leica cryostat, UK), and slides stored at −20°C. Sections were permeabilised with 0.5% Triton X-100 in PBS for 10 min at room temperature, washed in PBS and blocked with 10% goat serum (ThermoFisher Scientific) containing 1% bovine serum albumin (Sigma Aldrich) in tris buffered saline (TBS, Sigma Aldrich) for 2 h at RT. They were then incubated at 4°C overnight with primary antibodies anti fibronectin (1:100; ab2413 Abcam) collagen I (1:200; ab90395 Abcam), collagen IV (1:200; ab6586 Abcam), laminin (1:200; ab11575 Abcam) or KI67 (1:100; 14-5698-82 eBioscience). Sections were washed twice in 0.025% Triton-X (Sigma Aldrich) in TBS, before being incubated with secondary antibodies for 1 h at RT: goat-anti-rabbit (1:500; a11037 ThermoFisher Scientific), goat-anti-mouse (1:500; a11032 ThermoFisher Scientific) and goat-anti-rat (1:500; a11006 ThermoFischer Scientific). Stained samples were imaged using an Olympus IX70 inverted fluorescence microscope or Hamamatsu Nanozoomer S60 digital slide scanner.