Alexa fluor 488 conjugated 10 kda dextran

Macromolecular permeability of the endothelium in vascularized tissue was measured using a previous method [22 (link)]. Briefly, after 3–4 days of perfusion, media in the upper reservoir was replaced with perfusion media that was supplemented with 50 μg/mL Alexa Fluor 594-conjugated bovine serum albumin (BSA) and 20 μg/mL Alexa Fluor 488-conjugated 10 kDa dextran (both from Invitrogen). The tissue was placed in an environmental chamber at 37°C, and time-lapse images were captured every minute for thirty minutes in both red and green channels. Average gray values for the time-lapse images were measured using ImageJ and recorded. Permeability coefficients were determined using an algorithm to correct for the non-instantaneous filling of the vessel by fluorescent media [23 (link)].

To assess the viability of the endothelium, we perfused seeded channels with 50 μg/mL calcein AM (Invitrogen) and 10 μg/mL Hoechst 33342 (Invitrogen). To assess the patency of the endothelium, we perfused channels with media that contained 100 μg/mL Alexa Fluor 488-conjugated 10 kDa dextran (Invitrogen). Channels were perfused for 30-60 minutes under the same pressure and cAMP conditions tested experimentally. Fluorescence images were obtained with a Plan-Neo 10×/0.30 NA objective. Composite images were assembled in ImageJ ver. 1.43u (NIH) and Adobe Photoshop 6.0.