Fig. 3

eCIRP compromises cell-to-cell junctional integrity during neutrophil TEM. A The TEM assay was performed with either PBS or eCIRP (0.5 µg/mL) for 4 h and transwell membranes were prepared to do the immunofluorescence staining. Attached neutrophils and endothelial cells were directly stained on the transwell membrane with VE-cadherin antibody (red color) and mounted onto a glass slide for microscopy. Neutrophils (BMN) were pre-stained with Celltrace Violet (blue color) prior to the assay. Endothelial cells were stained with fluorescent membrane staining dye, Membrite Green (green color) at the beginning of the TEM assay. Bright field (BF) panel of the images show the pores (black holes; 3 µm in diameter pores) of the transwell membrane. Asterisks (*) in the image for eCIRP treated indicate the regions of transwell membrane, where the endothelial cells are void due to their migration to the bottom of the transwell membrane. Scale bar, 20 µm. B The VE-cadherin immunofluorescence was further analyzed by measuring the total area of VE-cadherin in the junctions in 5 different microscopic fields. The data obtained was normalized to the level of PBS treated sample. Data represented as mean ± SEM. *p < 0.05, unpaired, two-tailed student’s t-test