Axio imager m2 optical microscope

To label mitochondria, cells cultured on Ti-Control and Ti-Nano for 24 ​h before and after centrifugation were incubated with MitoTracker® probes (200 ​nM) for 45 ​min at 37 ​°C in a humidified atmosphere with 5% CO₂. Then, cells were washed with α-MEM without FBS followed by fixation for 15 ​min at 37 ​°C using 4% paraformaldehyde in PB. The samples were washed three times (5 ​min each) in PB. Glass slides were used to mount the discs face up, and cell nuclei were stained and mounted with mounting medium containing DAPI (Prolong antifade 4′,6-diamidino-2-phenyl-indole, dihydrochloride, Molecular Probes, Invitrogen) covered with round-glass coverslips. Images were acquired using a 63× objective with a Zeiss Axio Imager M2 Optical Microscope. More than 10 ​cells from three different replicates were analyzed by each condition.

Cells were visualized using an Axio Imager M2 optical microscope (Carl Zeiss Microscopy GmbH, Jena, Germany) in phase-contrast and fluorescent modes. The images were captured using cameras such as Axoicam 506 Color and Zen Blue 3.1 (Carl Zeiss Microscopy GmbH, Jena, Germany). The morphometric parameters of bacterial cells were investigated using a unique combined scanning system consisting of an Olympus FV 1000 confocal laser scanning microscope (CLSM) (Olympus Corporation, Tokyo, Japan) and an Asylum MFP-3D atomic force microscope (AFM) (Asylum Research, Santa Barbara, CA, USA). Sample preparation and AFM scanning procedure were carried out according to Kuyukina et al. [31 (link)]. For that purpose, the cell suspension (15–20 µL) was stained with a two-component fluorescent dye LIVE/DEAD^®BacLight^TM Bacterial Viability Kit (Invitrogen, Carlsbad, CA, USA). AFM scanning of the preparations was performed in a semi-contact mode in air at frequency of 0.2 Hz using an AC240TS silicon cantilever with resonance frequency of 50–90 kHz, spring constant of 0.5−4.4 N/m, and the curvature radius of the probe at 9 nm. The root-mean-square surface roughness and dimensions (length and width) of living cells were calculated using the Igor Pro 6.22A (WaveMetrics, Portland, OR, USA) software. Cell volume and surface area were calculated using the formulas in [32 (link)].

Apoptosis was studied with the TUNEL technique (Terminal deoxynucleotidyl transferase dUTP nick end labeling) [18 (link)]. 3 μm cuts were made from paraffin blocks of placental tissue, and Superfrost Plus^™ Microscope Slides (Thermo Scientific) were prepared. The tissue was deparaffinized with xylol and ethanol at different concentrations and subsequently paraformaldehyde to fix it to the sheet.
The DeadEnd^™ Colorimetric TUNEL System reagent set (Promega) was used for the colorimetric detection of apoptotic cells in the tissue fixed on the slide. The slides were viewed with a Zeiss Axio Imager M2 optical microscope equipped with a Zeiss AxioCam HRc camera, which was used to capture images of the placenta. The number of positive cells, which were a brown color, was calculated with the Image J program (J Image 1.46r Wayne Rasband National Institute of Health, USA, https://imagej.nih.gov/ij/index.html).
The percentage ratio of apoptotic cells was obtained by taking into account the number of positive nuclei (colored brown) and the number of total cells observed in 40 fields with a 40X objective; The number of TUNEL positive cells was divided by the total cells counted and multiplied by 100 to find the percentage.

To detect the effect of the hierarchical micro/nano topography on mature FA formation, wild‐type cells, scramble cells and Rictor knock‐down cells were seeded at a density of 1 × 10³ cell/well on three different surfaces for 24 hours. Vinculin was employed to represent FA, and was stained according to the protocol of immunofluorescence described above. Confocal images were observed by using Zeiss Axio Imager M2 Optical Microscope (Carl Zeiss, Germany). The mature FA was defined as in the previous study we have published.¹⁶ Briefly, Image J software was employed to calculate the area of vinculin stain. The area which was greater than 3.14 µm² was acted as mature FA.

Sections were rehydrated as previously described then blocked using 2% BSA in TBS-T for 1 h at room temperature (Lévesque et al., 2010 (link)). Primary antibody was diluted in blocking solution (anti-p-Erk1/2, cat# 4370, Cell Signaling Technology, 1/400) and incubated on a slide overnight at 4°C. Secondary HRP coupled antibody was diluted (anti-rabbit HRP, cat# 170-6515, Bio-Rad, 1/800) in blocking solution for p-Erk1/2 and incubated at room temperature for 45 min. Tyramide (Biotium, San Francisco Bay, CA, cat# 92175) was diluted in 1× TBS with 0.0015% H₂O₂ to an active concentration of 11.6 µM then incubated at room temperature for 8 min. All slides were mounted with ProLong^® Gold antifade reagent containing DAPI (Invitrogen, cat# 36931). Slides were visualized with a Zeiss Axio Imager M2 Optical Microscope (Zeiss, Munich, Germany). The software used was the Zen 2 Pro Blue Edition (Zeiss, Munich, Germany, https://www.zeiss.com/microscopy/int/products/microscope-software/zen.html) with a Tile Module. All photos were verified using the range indicator of the software to make sure that they were not saturated. The photos were saved as TIF files and then imported into Photoshop CS4 (Adobe) to adjust the rotation and to crop the photos to be mounted into a multipanel figure using Illustrator CS4 (Adobe).