Goat anti mouse alexa fluor plus 488 secondary antibody

Substrates with collagen coating are blocked with 1% bovine serum albumin (Sigma, MO, USA) in PBS for 1 hour, followed by a two-step immunostaining process. Briefly, samples are first incubated with mouse monoclonal anti-collagen I antibodies (ab90395, Abcam, MA, USA) diluted 200X in PBS with a supplement of 1% bovine serum albumin for 1 hour at room temperature. Samples are then washed 5 times with PBS and incubated with goat anti-mouse Alexa fluor plus 488 secondary antibodies (Thermo Fisher Scientific Inc, MA, USA) diluted 200X in PBS with a supplement of 1% bovine serum albumin for 1 hour in the dark. Samples are washed 3 times with PBS before imaging. Substrate without collagen coating is stained with the same protocol as a negative control. The stained samples are then fluorescently imaged with a confocal microscope equipped with a 25X/0.95-NA water immersion objective (TCS-SP5; Leica Microsystems Inc., IL, USA).

Cells are seeded on substrates at a density of ~4000 cells/cm² and cultured in an incubator infused with 5% CO₂ and maintained at 37 °C. After 16 hours, cells are fixed with 4% formaldehyde and 0.1% Triton X100 diluted in PBS, followed by PBS wash 3 times to remove excessive reagents. Fixed cells are then triple stained for actin, vinculin, and nucleus: fixed cells are blocked with 10% normal goat serum (Thermo Fisher Scientific Inc, MA, USA) in PBS for 1 hour, followed by a two-step immunostaining process for vinculin. Briefly, cells are first incubated with mouse monoclonal anti-vinculin antibodies (Sigma-Aldrich, MO, USA) diluted 200X in PBS with a supplement of 10% normal goat serum for 1 hour at room temperature. Samples are then washed 5 times with PBS and incubated with goat anti-mouse Alexa fluor plus 488 secondary antibodies (Thermo Fisher Scientific Inc, MA, USA) diluted 200X in PBS with a supplement of 10% normal goat serum for 1 hour in the dark. Phalloidin-iFluor 555 (Abcam, MA, USA) and Draq 5 nucleus probe (Thermo Fisher Scientific Inc, MA, USA) are diluted at ratios of 1:1000 and 1:5000 each to stain actin and nuclei of cells. Stained cells are washed 3 times with PBS and imaged with a confocal microscope equipped with a 63X/1.20-NA water immersion objective (TCS-SP5; Leica Microsystems Inc., IL, USA).