eclipse ti2 spinning disk confocal microscopy system  (Nikon)

Journal: Advanced Science

Article Title: Engineered Biomimetic Fibrillar Fibronectin Matrices Regulate Cell Adhesion Initiation, Migration, and Proliferation via α 5 β 1 Integrin and Syndecan‐4 Crosstalk

doi: 10.1002/advs.202300812

Figure Lengend Snippet: Engineering 2.5D and 3D fibrillar fibronectin (FN) matrices. a) Schematic illustration of engineering 3D fibrillar FN matrices and 2.5D fibrillar FN matrices. First, a microporous grid (500 × 500 µm 2 square pores, 200 µm thickness and a size of ≈6 × 6 mm 2 ) made from an ABS‐like resin is 3D printed. The grid is placed in an Eppendorf tube filled with full‐length FN in PBS. The tube is then rotated (25 rpm) at 30 °C for 2 h to apply hydrodynamic shear force between the microgrid, FN solution, and air. After rotation, the grid embedded with a 3D fibrillar FN matrix can be removed from the Eppendorf tube and used. Contacting printing 3D fibrillar FN matrices onto glass covers the inorganic substrate with 2.5D fibrillar FN matrices. b) Representative fluorescence images of 2D globular FN substrates, 2.5D fibrillar FN matrices, and 3D fibrillar FN matrices. Fluorescence images show full‐length FN (green), unfolded FN (FN IST‐9, red), and both images merged. Dashed boxes in 2D globular FN substrates indicate the bleached area to distinguish fluorescent signal from background. Scale bars, 20 µm. c) AFM topography of 2D globular FN substrates scratched in the middle square area (purple dashed square). The height profile (red line) is generated along the red line in the topography. Scale bar, 5 µm. d) XZ projection confocal images of fibrillar FN matrices (green, full‐length FN antibody). Scale bar, 10 µm. e) Thickness analysis of globular FN substrates and fibrillar FN matrices. The mean thickness is 6.50 ± 0.31 nm for 2D globular FN substrates, 23.82 ± 4.93 µm for 2.5D fibrillar FN matrices, and 94.12 ± 9.59 µm for 3D fibrillar FN matrices. Dots represent the number of samples analyzed. Red bars indicate the mean and orange bars the standard error of the mean (s.e.). f) 3D reconstruction of confocal images showing a large‐scale coverage of fibrillar FN matrices across the 3D microporous grid. g) Engineered 3D fibrillar FN matrices are insoluble in DOC. Engineered 3D fibrillar FN matrices were treated with deoxycholate (DOC) solution (1% w/v) in PBS for a week. Confocal images of fluorescently stained (full‐length FN antibody, green) FN matrices were recorded before and 1 week after DOC treatment. Scale bars, 50 µm. h) Fluorescence image of fibrillar FN matrices (FN IST‐9, red) deposited by fibroblasts in vitro. Scale bar, 50 µm. i) Representative scanning electron microscopy (SEM) images of 2D globular FN substrates, 2.5D fibrillar FN matrices, and 3D fibrillar matrices. Scale bars, 1 µm.

Article Snippet: Engineered FN substrates were incubated with anti‐FN (full‐length FN, Abcam, ab2413) and FN IST‐9 (Abcam, ab6328) with 1:100 dilution in PBS at room temperature for 1 h. After incubation, substrates were washed twice by PBS and incubated with Alexa Fluor 488 goat anti‐rabbit IgG (for full‐length FN) and Alexa Fluor 594 goat anti‐mouse IgG (for FN IST‐9) antibodies at room temperature for 1 h. After incubation, substrates were washed twice by PBS and imaged using an inverted spinning disk confocal microscopy with 40× objective (W1‐SoRa Eclipse Ti2‐E, Nikon).

Techniques: Shear, Fluorescence, Generated, Staining, In Vitro, Electron Microscopy