Rabbit polyclonal anti laminin

After 28 days of culture, Unpass-ASC were fixed with PFA 1% for 1 hour and washed twice with PBS. Immunohistochemical staining using antibodies against Collagen type I (MP Biomedicals, Illkirch-Graffenstaden, France) and fibronectin was performed as previously described8 (link). After incubation with a biotinylated secondary antibody and subsequently with an ABC-alkaline phosphatase complex, the specific staining was revealed by using Fast Red (All reagents from Dako, Baar, Switzerland). Matched IgG control antibodies were used as negative control.
For immunofluorescent staining, freshly harvested human fat tissue was frozen in liquid nitrogen, fixed in pure acetone for 10 minutes at −20 °C and then cryosectioned. Sections of 10 μm in thickness were stained with the following primary antibodies and dilutions: mouse anti-human CD49e (BD Biosciences, Basel, Switzerland) at 1:50; mouse anti-human; rabbit monoclonal anti-fibronectin (Abcam, Cambridge, UK) at 1:200; and rabbit polyclonal anti-laminin (Abcam) at 1:50. Fluorescent-conjugated secondary antibodies (Invitrogen, Basel, Switzerland) were used at 1:200. Images were acquired with a Nikon A1R confocal microscope.

ECs cultured in microfluidic devices were fixed by adding 4% (w/v) paraformaldehyde (in PBS) into each reservoir of the devices and incubating for 15 minutes. Cells were permeabilized with 1% Triton-X100 (Sigma-Aldrich) in PBS for 10 minutes. After blocking with 20% Block Ace (in PBS) (Dainihon-Seiyaku, Japan) for 1 hour and washing the channels with PBS, solutions of rabbit polyclonal anti-laminin (1:100; Abcam, USA), rabbit polyclonal anti-CLDN1 (5 μg/mL; Abcam), rabbit polyclonal anti-CLDN5 (1:100; Abcam), rabbit polyclonal anti-OCLN (2 μg/mL; Abcam), and mouse monoclonal anti-integrin alpha 6 (1 μg/mL; Abcam) primary antibodies were introduced into the devices and incubated for 1 hour at room temperature. After washing with PBS, Alexa Fluor 488-conjugated goat anti-rabbit and goat anti-mouse IgG secondary antibodies (1:200; Molecular Probes, USA) were added to the device and incubated for 1 hour. Cell nuclei and actin filaments were counterstained with 4′, 6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich) and rhodamine phalloidin (Sigma-Aldrich), respectively. Stained cells were observed under a fluorescence microscope (Axio Observer D1; Carl Zeiss, Germany) and a confocal microscope (LSM-700; Carl Zeiss).