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. Author manuscript; available in PMC: 2011 Aug 1.
Published in final edited form as: J Immunol. 2010 Jun 25;185(3):1878–1886. doi: 10.4049/jimmunol.1001305

Figure 5. Leukocyte PECAM moves to a Triton insoluble fraction during transmigration.

Figure 5

(A) HUVEC monolayers labeled with AlexaFluor488 conjugated anti-VE-cadherin and human monocytes adhered to glass labeled with non-blocking AlexaFluor546 anti-PECAM with or without addition of secondary cross-linking antibodies. Samples were fixed in 2% paraformaldehyde or incubated with a Triton X100 extraction buffer at 4°C for 30minutes before fixation and imaged by wide-field microscopy. (B) Cells were labeled as in A and monocytes were added to endothelial monolayers for 10 minutes at 37°C. Cells were then processed and fixed as above. Leukocytes were seen adhered away from endothelial junctions (asterisks) and directly over junctions (arrows). Representative images are shown. (C) The maximal fluorescent intensity of PECAM found on leukocytes at junction and non-junction positions was analysed for 40 cells. Graphs show individual data points with mean fluorescent intensity, **P<0.007.