Rabbit polyclonal antibody against laminin

Cells and extracellular matrix were visualized using immunofluorescence staining. Briefly, after washing with PBS, the microvasculature in the PDMS device was fixed with 4% (w/w) paraformaldehyde in PBS for 30 min and then permeabilized with 0.5% Triton-X in PBS for 10 min. Blocking was performed using 1% BSA in PBS overnight at 4 °C. For staining F-actin, the cells were exposed to Alexa Fluor 488-phalloidin at 25 °C for 2 h, and for nucleus, Hoechst 33342 at 25 °C for 15 min. For staining laminin and podocalyxin, after blocking, the microvasculature was incubated with rabbit polyclonal antibody against laminin (1:100; ABcam) and goat polyclonal anti-human podocalyxin (1:100; R&D) overnight at 4 °C. The microvasculature was then washed well with PBS and exposed to Alexa Fluor 568-secondary antibodies and Alexa Fluor 488-phalloidin together. Both anti-rabbit and anti-goat secondary antibodies (Invitrogen) were used at 1:100 dilutions. Images of the stained 3D microvasculature were obtained using a confocal microscope (LSM700; Zeiss, Oberkochen, Germany) with lenses of 10 × magnification. Confocal images were processed using the software ZEN version 8.1 (Zeiss) to construct 3D projection images from Z-stack images.