Hoechst 33342 h3570

For the immunostaining performed for stretching and toxicity analysis, samples were fixed in 2% PFA, permeabilized with 0.5% Triton X-100, and blocked with 5% FBS at room temperature. Primary antibody TUBB3 (#T8578, 1:500, Sigma, Darmstadt, Germany) was diluted in 3% FBS/0.2% Triton X-100 in DPBS and incubated overnight at 4 °C. Samples were then washed and incubated with secondary antibody (R6393, 1:500, Invitrogen, Carlsbad, CA, USA) and Hoechst 33342 (H3570, 1:1000, Invitrogen, Carlsbad, CA, USA). All images were acquired using a fluorescent microscope (TE2000-U, Nikon, Minato, Tokyo, Japan).
For the immunostaining performed on 2-well silicone insert, cells were fixed in 2% PFA and 7.5% sucrose, permeabilize in 0.1% Triton X-100 and blocked with 5% goat serum (GS) at room temperature. Primary antibodies TUBB3 (#T8578, 1:500, Sigma, Darmstadt, Germany) and KDEL (PA1-013, 1:200, Thermo Scientific, Waltham, MA, USA) were incubated overnight at 4 °C. Samples were then washed and incubated with secondary antibody (#A21449 and #06380, 1:500, Thermo Scientific, Waltham, MA, USA) Hoechst 33342 (H3570, 1:1000, Invitrogen, Carlsbad, CA, USA). All images were acquired using a laser scanning confocal microscope (Eclipse Ti, Nikon, Minato, Tokyo, Japan).

All chemicals used were of analytical reagent grade. Milli-Q water was used for the preparation of standards and reagents. DAPI (23018) was purchased from Biotium (Fremont, CA, USA). Hoechst33342 (H3570) was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Antibody to fibronectin (P1H11) was obtained from the Developmental Studies Hybridoma Bank (DSHB, Iowa City, IA, USA). Fibronectin (Ab268020) and tenascin C (ab108930) antibodies were purchased from Abcam (Cambridge, MA, USA). αSMA antibody (F3777) was purchased from Millipore-Sigma (St. Louis, MO, USA). N-cadherin antibody (14215) was purchased from Cell Signaling Technology (Danvers, MA, USA). Cadherin-11 antibody (368702) was purchased from BioLegend (San Diego, CA, USA). ZO-1 (40-2200) and GAPDH (PA1-987) antibodies were purchased from Thermo Fisher Scientific. The Cell Counting Kit-8 assay kit (HY-K0301) was purchased from MedChem Express (Monmouth Junction, NJ, USA). All other chemicals were obtained from Sigma-Aldrich and Thermo Fisher Scientific.