Imaris 64

For SBF-SEM an image stack of a bovine placenta sample was used. The sample was collected from a placentome of a pregnant Holstein Friesian cow (gd 278 days), terminated by cesarean section, which was performed after rupture of fetal membranes. Preservation in Karnovsky’s fixative was followed by embedding in Durcupan’s resin, as previously described [56 ]. These were recorded with a section thickness of 60 nm. SBF-SEM images were used for further processing in 3D reconstruction and modeling of the target cell type.
ROIs with well-preserved areas of maternal stroma were selected on single SEM surface scans of the block. The selected image field, showing the target cells, was utilized for the creation of an image stack with 970 images. 3D reconstruction was performed on a defined stack of 180 images (Voxel size = 15 × 15 × 60 nm), which included a complete myofibroblast. Herewith, a manual segmentation of the nucleus, the stress fibers and the cytoplasm, was conducted, using the ImageJ software (ImageJ 1.53q, bundled with 64-bit Java, version 1.8.0_172) plugin TrakEM2 [57 (link)]. For visualization, segmented images were merged and resulting channels were used to create a 3D model (Imaris ×64, version 9.9.1, Bitplane AG).

PBSe-GSK3787-NR particles were resuspended in culture media at a concentration of 100 μg/mL, then sterilized under the UV light of the cell culture hood for 1 h. Then, 1 mL of the particle containing media was added to each cell containing well in the 24 well plate, and then the cells were incubated for 48 h. The media was then aspirated, and the cells were washed 3 times with PBS before being fixed with a 4 wt.% paraformaldehyde (PFA) solution for 10 min at room temperature. After washing with PBS, 1 wt.% Triton X-100 was added and cells were incubated for 10 min at room temperature. Cells were washed with PBS again before adding a 1% bovine serum albumin (BSA) solution and incubating at room temperature for 30 min. AlexaFluor 488 Phalloidin stain was added to PBS at a concentration of 10 μg/mL, then 1 mL of the PBS containing AlexaFluor 488 was added to cells and incubated for 10 min at room temperature. Coverslips were washed with PBS before being removed and fixed to glass slides using Immunomount with DAPI. Slides were stored in the dark until imaging. Confocal microscopy was performed using a Zeiss LSM 900 confocal microscope. A 3D rendering of confocal images was created using Oxford Instruments Imaris ×64 software.