Podocalyxin

Pre-implantation embryos were fixed and stained as previously described (Bedzhov et al., 2012 ). Peri-implantation embryos were fixed with 4% PFA/PBS for 20 min. Cellular permeabilization was carried out for 10 min in 0.1M Glycin, 0.3% Triton X-100/PBS. The embryos were in incubated in primary antibody in 10% FCS/PBT for 2h to overnight at room temperature. Secondary antibodies were applied subsequently for 2h. Embryos were stained with DAPI (Invitrogen) and mounted in PBS droplets covered with mineral oil in glass bottom petri dishes. Confocal microscopy was performed using a Leica SP5 microscope and the images were processed using Imaris software (Bitplane). Antibodies: cleaved Caspase-3 (1:200, Cell signaling, 9664), Oct4 (1:200, Santa Cruz, sc-5279), Sox2 (1:200, Santa Cruz, sc-17320), aPKC (1:200, Santa Cruz, sc-216), Par6 (1:200, Santa Cruz, sc-67393), E-cad (1:200, (Vestweber and Kemler, 1984 )), gm130 (1:200, Calbiochem, CB1008), Eomes (1:200, Abcam), Nanog (1:200, Abcam, ab80892), Laminin (1:500, Sigma, L9393), Sox17 (1:200, R&D systems, AF1924), pMLC (1:200,Cell signaling, 3674), podocalyxin (1:200, R&D systems, MAB1556), β1-integrin (1:200, BD Pharmigen, 555005).

Immunofluorescence staining was performed on cell monolayer, 2 hours after the scratch/wounds was performed. Cells were fixed in 4% paraformaldehyde (Electron Microscopy Sciences) at room temperature for 15 minutes and permeabilized in 0.1% Triton X-100 (Sigma) for 5 minutes. After blocking with 5% horse serum (Vector Laboratories) for 1 hour, cells were incubated with Alexa Fluor® 488 mouse anti-GM130 antibody (12.5 μg/ml; BD Biosciences) or ICAM2 (0.56 μg/ml; Cell Signaling) for 1 hour. Filamentous actin was stained using Phalloidin conjugated to the fluorescent dye tetramethylrhodamine (TRITC) (1:40; Life Technologies). Samples were mounted using Prolong Gold with 4′,6-diamidino-2-phenylindole (DAPI) (Life Technologies) and imaged with a C2 confocal microscope (Nikon).
Whole mount staining was performed on fibrin gels upon overnight fixation in 4% paraformaldehyde at 4 °C. Immunofluorescence was performed using primary antibodies against Podocalyxin (10 μg/ml; R&D Systems), Collagen IV (5 μg/ml; eBioscience) followed by Texas Red and FITC -conjugated secondary antibodies (1:200, Vector Laboratories). Nuclei were stained with DAPI (4′,6-diamidino-2-phenylindole) (1:1000, Life Technologies) and imaged Nikon A1R LUN-V Inverted Microscope.

Immunofluorescence staining was performed by following the method described previously [53 (link)]. Briefly, kidneys were embedded in optimal cutting temperature compound (Sakura Finetek, Tokyo, Japan), and frozen in liquid nitrogen. Sections of 5-μm thickness were cut by a cryostat (CM3050; Leica, Wetzlar, Germany); then incubated with primary antibodies against type I collagen (Acris Antibodies, Germany), type IV collagen, type V collagen, synaptopodin, VE-cadherin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), nephrin, and podocalyxin (R&D Systems, Minneapolis, MN, USA), respectively, at 4 °C overnight, and followed by secondary antibodies conjugated to Alexa Fluor^® 488 or 568 (Invitrogen, Carlsbad, CA, USA); double-stained with rhodamine-conjugated phalloidin (Life Technologies, Gaithersburg, MD, USA) for F-actin; and finally submerged in fluoroshield mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI) (Abcam, Cambridge, UK). Confocal imaging was performed according to the method described previously [26 (link)] with a confocal microscope (LSM700; Carl Zeiss, Jena, Germany). Alexa Fluor^® 488, and 568 signals were detected at laser excitation wavelengths of 488 nm and 543 nm, respectively.