Ab196579

Cells were washed three times with ×1 PBS and then fixed with 4% paraformaldehyde (PFA) for 12 min. After being penetrated by Triton X-100 (0.25%–0.5%) for 5 min, the cells were blocked by bovine serum albumin (BSA, 5%) for 2 h. Cells were incubated with antibodies, e.g., phalloidin (FITC, A12379, Thermo) and vinculin (ab196579, Abcam, Cambridge, UK) antibodies. Then, 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (Dapi, D9542, Sigma, St. Louis, MO) was applied to stain the nuclei for 10 min at 10 μg·mL⁻¹. The immunofluorescence images of cell samples were observed through a confocal laser scanning microscope (FV3000, Olympus, Tokyo, Japan and A1R MP+, Nikon, Tokyo, Japan, and parameter: 40, Nikon Microsystems original image: 1 024 × 1 024). The images presented in the figures are representative of a projection of all slices based on at least three independent experiments (n = 3). For the quantification of immunofluorescence intensity, we used the connected software in FV3000, Olympus and A1R MP+, Nikon.

Formation
and distribution of focal adhesions in hMSC-nanofunctionalized microparticle
spheroids were qualitatively assessed via confocal fluorescence microscopy
analysis. After 4 and 24 h in culture, the spheroids were fixed with
warm 10% FA in DPBS solution for 20 min and permeabilized using a
0.1% v/v solution of Triton-X100 (Sigma-Aldrich) in Milli-Q water
for 30 min. After this, an unspecific binding blocking step of 1 h
was performed in a casein-based blocking serum (CAS-Block Histochemical
Reagent; Thermo Fisher Scientific). The spheroids were then incubated
overnight with an Alexa Fluor 647-conjugated monoclonal antivinculin
antibody (ab196579, Abcam) solution in a 0.1% w/v bovine serum albumin
(BSA; VWR) solution in phosphate-buffered saline (PBS) with a 1:100
dilution to visualize focal adhesions. At the same time, cell nuclei
were counterstained overnight using diamidino-2-phenylindole dihydrochloride
(DAPI; Sigma-Aldrich) at a dilution of 1:200 in 0.1% BSA in PBS. The
imaging was performed using a confocal laser scanning electron microscope
(TCS SP8 STED, Leica Microsystems), acquiring z-stacks of 4 μm
thickness for each image.