Metavue 6.2r2 imaging software

At the end of the experiments silicone membranes with confluent monolayers were stained with specific antibodies for NFKB p50/p105 (Santacruz, California), NFKB p65 (Cell Signaling, Massachusetts) and CDH5 and visualized using fluorescence microscopy. Silicone membranes were washed twice with PBS and fixed in 1% PFA and 4% PFA for NFKB p65 for 10 min, cut into sections, and permeabilized with Triton X-100 in PBS for 10 min. After permeabilization samples were blocked in 10% BSA and 0.1% Triton X-100 in PBS (NFKB and CDH5) for 1h. After washing with 0.1% Triton X-100 in PBS, the silicone sheets were incubated with polyclonal rabbit anti- NFKB, and CDH5 primary antibodies overnight at 4°C followed by washing with 0.1% Triton X-100 in PBS. Samples were subjected to secondary antibody Alexa Fluor 488 donkey anti-rabbit (1:500 to 1:200; Invitrogen) secondary antibody for 1.5 h for CDH5 and with secondary antibody Alexa Fluor 488 donkey anti-goat (1:100 to 400; Santa Cruz) for 1.5h for NFKB. Samples were washed again with 0.1% Triton X-100 in PBS and mounted with vectashield mounting media with DAPI on glass slides with cover slips in contact with cells. These slides were imaged using a Nikon Eclipse TE2000-E inverted fluorescence microscope with a Photometrics Cascade 650 camera (Roper Scientific) and MetaVue 6.2r2 imaging software (Universal Imaging).

A specific antibody for NFkB p65 (Santacruz, California) was visualized using fluorescence microscopy. Cell-laden silicone membranes were washed twice with PBS and fixed in 1% PFA for 10 min, cut into sections, and permeabilized with Triton X-100 in PBS for 10 min. After permeabilization samples were blocked in 10% BSA and 0.1% Triton X-100 in PBS for 1h. Then the silicone sheets were incubated with polyclonal rabbit anti- NFkB primary antibodies overnight at 4°C followed by washing with 0.1% Triton X-100 in PBS. Samples were subjected to secondary antibody Alexa Fluor 488 donkey anti-goat (1:100 to 400; Santa Cruz) for 1.5h for NFκB. Samples were washed again with 0.1% Triton X-100 in PBS and mounted with vectashield mounting media with DAPI on glass slides with cover slips in contact with cells. These slides were imaged using a Nikon Eclipse TE2000-E inverted fluorescence microscope with a Photometrics Cascade 650 camera (Roper Scientific) and MetaVue 6.2r2 imaging software (Universal Imaging).