For IF of 3D-cultured ReN cells, thin-layer 3D cultures were fixed with 4% PFA at room temperature for 24 hours. The fixed cells were then permeabilized and blocked by incubating with a blocking solution containing 50 mM Tris (pH 7.4), 0.1% Tween-20, 4% donkey serum, 1% BSA, 0.1% gelatin and 0.3 M glycine at 4°C for 12 hours. After washing with TBS buffer containing 0.1% (v/v) Tween-20 (TBST), the 3D cultures were incubated with primary antibodies in the blocking solution at 4°C for 24 hours. After washing 3 times with TBST, the cells were then incubated with TBST overnight by gentle rocking at 4°C and then further incubated with AlexaFluor secondary antibodies (Life Technologies) overnight at 4°C. To avoid fluorescence quenching, a drop of anti-fade gold (Life Technologies) was added on top of the fixed/stained thin-layer 3D cultures before imaging. The fluorescence images were captured by Olympus DSU confocal microscope (Olympus USA, Center Valley, PA, USA) and the image analysis and 3D reconstitution were performed with ImageJ (a public domain image analysis software), IPlabs (BioVision Technologies, Exton, PA, USA) and
