Rabbit anti fn

Samples were fixed after 4 or 24 h with 4% (w/v) paraformaldehyde (Merck, #1040051000). A standard staining procedure was performed with the following antibodies and dyes (final concentration): Rabbit anti-FN (1.2 µg/ml, Sigma, #F3648), rabbit anti-LN (5 µg/ml, Sigma, #L9393), mouse anti-YAP (0.33 µg/ml, Santa Cruz, #sc-101199; applied at 4 °C over night), goat anti-mouse Cy3 (7.5 µg/ml, Jackson ImmunoResearch, #115-165-166), goat anti-rabbit AF568 (7.5 µg/ml, Jackson ImmunoResearch, #111-165-144), goat anti-rabbit AlexaFluor (AF) 647 (7.5 µg/ml, Jackson ImmunoResearch, #111-606-045), DAPI (2–0.2 µg/ml, Roth, #6335.1), Phalloidin AF568 (1 U/ml, Invitrogen, #A12380), Phalloidin AF647 (2 U/ml, Invitrogen, #A22287).

En face mounted tissue samples were rehydrated with water for 10 minutes. The tissue was then permeabilized using Tris-Triton buffer (50 mM Tris, 500 mM NaCl, 0.3% Triton X-100) for 1 hour, followed by 3 washes with water. Permeabilized tissue was then blocked using 0.5% non-fat dry milk and 1% BSA in TBST for 60 minutes at room temperature. Primary antibodies (Rabbit anti-FN, 1:100, Sigma F3648, St. Louis, MO; rat anti-VE-Cadherin, 1:50, BD Pharmingen 550548, Franklin Lakes, NJ,; or rabbit anti-Collagen α1, 1:100, Abcam Ab292, Cambridge, UK) were then diluted in blocking buffer and incubated with the tissue overnight at 4 °C. After primary antibody incubation, slides were washed 3 times with TBST. Secondary antibodies (Goat anti-rat Alexafluor 488, 1:100, A11006 Life Technologies; Goat anti-rabbit Alexafluor 568, 1:100, A11011 Life Technologies) were diluted in blocking buffer and incubated with the tissue for 60 minutes at room temperature. The tissue was washed 3 times in TBST, and then mounted with VectaShield plus DAPI (Vector Labs, Burlingame, CA) and sealed with clear nail polish. Slides were imaged on a Zeiss 700 or Zeiss 710 confocal microscope.