The cultured cells on coverslips were fixed in 4% formaldehyde, permeabilized by incubation in 0.5% Triton X-100, blocked with 10% goat serum, and then stained with polyclonal anti-fibronectin (Sigma-Aldrich) followed by Alexa-647 conjugated goat anti-rabbit antibody (Molecular Probes/Invitrogen). Afterwards, the samples were stained for f-actin, g-actin by Alexa Fluor® 488 phalloidin and the nuclei by DAPI (Molecular Probes/Invitrogen). To assess the presence of platelets in the colonic mucosa, sectioned colonic tissue mounted on the slides were stained with a rat anti-CD41 antibody, followed by a FITC conjugated goat anti-rat polyclonal antibody, and the nuclei by DAPI, based upon a previously described technique (31 (link), 32 (link)).
Finally, samples were mounted on slides with elvanol (DuPont). Specimens were then visualized either by Deconvolution Microscopy employing an Olympus IX-70 microscope connected to a DeltaVision imaging system (Applied Precision, Issaquah WA), or Nikon A1R confocal laser microscope system and the NIS-Elements C software. The quantitation of immunofluorescence density was done using ImageJ software by measuring pixel units.
Finally, samples were mounted on slides with elvanol (DuPont). Specimens were then visualized either by Deconvolution Microscopy employing an Olympus IX-70 microscope connected to a DeltaVision imaging system (Applied Precision, Issaquah WA), or Nikon A1R confocal laser microscope system and the NIS-Elements C software. The quantitation of immunofluorescence density was done using ImageJ software by measuring pixel units.