HCECs or HCLE cultured on different silk film substrates were fixed with 4% paraformaldehyde (PFA; Electron Microscopy Sciences, Hatfield, PA, USA) for 15 minutes, then rehydrated in PBS containing 0.5% bovine serum albumin (BSA; Sigma-Aldrich Corp.), and 0.05% nonionic surfactant (Triton-X-100; Sigma-Aldrich Corp.) for 1 hour. After fixation, 50 μL primary antibodies (1:600 antivinculin V9131, Sigma-Aldrich Corp.; 1:50 anti-integrin β 1, 12594-1-AP, Proteintech, Rosemont, IL, USA; 1:50 antinesprin 2, GTX121928; GeneTex, Irvine, CA, USA) was incubated with cells overnight at 4°C, followed by incubation with Oregon Green 488 goat antimouse secondary antibody (O11033; Invitrogen Corporation) or Cy3 goat antirabbit secondary antibody (711165151; Jackson Immunoresearch, West Grove, PA, USA) at a 1:500 dilution for 1 hour. F-actin and nuclei were then stained by incubating cells in 1:100 dilution of Alexa Fluor 568 Phalloidin (A12380; Invitrogen Corporation), and 1:10,000 dilution of 4′,6-diamidino-2-phenylindole (DAPI) (83210, AnaSpec, San Jose, CA, USA) for 20 minutes and 5 minutes, respectively, while protected from light. After rinsing using PBS, samples were mounted, and protected with a glass cover slip. Fluorescent staining was visualized using Observer Z1 confocal fluorescent microscope (Carl Zeiss, AG) with both a 10× and 63× objective lenses. An AxioCam HRm digital camera (Carl Zeiss) and AxioVision 4.0 software were used to capture single and z-stack images (45- to 60-layer range) at 0.25-μm slices using DAPI, GFP, and Texas Red filter channels. Deconvolution was performed on each z-stack using 3D Huygens Deconvolution Software (Scientific Volume Imaging BV, The Netherlands). Images for each replicate were taken at the same threshold level. Total fluorescence measurements were performed with Image J software (National Institutes of Health).