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, Samples were then washed 3× for 15 min in PBX, and fluorescent dyecoupled secondary antibodies were incubated for 90 min in PBX and 1% BSA at RT. After several washes in PBX, the stained tissues were then transferred to CitiFluor AF1 (Agar) mounting media for overnight equilibration. Individual discs were then dissected and mounted in CitiFluor. Images were acquired with an SP2-405 confocal microscope (Leica Microsystems) or an Apotome2 microscope (ZEI SS) and processed using ImageJ, p.2
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:25; 4F3; DSHB), mouse anti-Ena (1:200; 5G2; DSHB), mouse anti-Rho1 (1:25; p1D9; DSHB) ,
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