Xc30 camera

Here, 4% formaldehyde in phosphate buffer solution (PBS) was used to fix the samples, which were dehydrated in a graded series of ethanol and embedded in paraffin blocks. Samples sections (5 μm) were deparaffinized, hydrated in a graded series of alcohol solutions, and stained with H&E for cell colonization analysis throughout the scaffolds and Alizarin Red S to visualize the calcium-rich cell matrix at T0, 7 days, and 28 days of osteogenic differentiation. Light microscopic images were taken with Olympus Bx 43 microscope (Olympus, Tokyo, Japan) equipped with Olympus XC30 camera.

For invasion assays, we used Corning^® BioCoat^™ Matrigel^® Invasion Chamber (Corning, Discovery Labware, Bedford, US). The collagen layer of the cell culture inserts was rehydrated by adding 500 μl of serum-free RPMI-1640 to the inner compartment, and 1 × 10⁵ cells were seeded inside of each insert in serum-free RPMI-1640 with or without TGFβ. The lower well of the invasion plate was filled with 500 μl RPMI supplemented with 10% FBS. After incubation for 24 hours, non-invasive cells were removed from the interior of the inserts. Invasive cells were photographed using an Olympus BX50 microscope with a 20 × /0.5 objective length after staining with Cell Stain Solution. The images were acquired using CellA software at room temperature, using an Olympus XC30 camera. Each insert was treated with 200 μl of Extraction Solution for 10 minutes. Optical density (OD) at 560 nm was measured in a plate reader.

Biopsies from control positive and grafted gastric defect were collected at the end of the experiment and fixed in 10% neutral buffered formalin for 48 h. These biopsies were then processed by the paraffin-embedding technique. Tissue sections 5 μm thick were prepared using microtome (Leica 2135) and stained with H&E stain for microscopic examination [34 ]. Re-epithelization and leukocytic cell infiltrate in induced ulcers were evaluated in control and AM-treated ulcers. Semiquantitative scores of lymphocytes from 0 to 3 were used for inflammation. Inflammation of the ulcerated region was scored by counting lymphocytes in three fields (200× magnification) in three observer-randomized H&E slides (semiquantitative score: 0, < 5% cells/field; 1, 5–25%; 2, 25–50%; 3, > 50%) [35 (link)].
Tissue sections from each paraffin-embedded block were also stained with Masson’s Trichrome to detect fibrosis [34 ]. The stained tissue sections were examined by light microscopy and photographed using an Olympus XC30 camera (Tokyo, Japan).

To determine the multipotency of MSC overexpressing FasL, cells transduced as described above and were seeded at a 5000 cell/cm² in 24-well plates and left to adhere overnight. Twenty-four hours after transduction, media was changed with adipogenic and osteogenic induction media, respectively, as well as normal MSC media. Media was changed every 2 to 3 days. The adipogenic induction media contained DMEM 1-g/L glucose supplemented with 10% MSC-FBS, 10⁻⁶-M dexamethasone, 100-mM indomethacin and 1% insulin-transferrin-selenium (ITS-G, ThermoFisher, Waltham, MA, USA), and the osteogenic induction media was based on DMEM 1-g/L glucose supplemented with 10% MSC-FBS, 10⁻⁷-M dexamethasone, 10-mM β-glycerophosphate and 0.3-mM ascorbic acid. After 2 weeks, cells were washed with phosphate buffer saline (PBS), fixed with 4% paraformaldehyde and stained with Oil Red O to visualize the lipid droplets accumulated in the differentiated adipocytes or with von Kossa stain represented by incubation in 5% AgNO₃, followed by a short 2-min rinse with 5% sodium thiosulphate to visualize the Ca²⁺ crystals. All the images were taken using an Olympus CKX41 microscope with an Olympus XC30 camera and minimally processed using Adobe Photoshop software.

The collected specimens obtained through biopsy were fixed in 10% neutral formalin buffer and then processed using a paraffin embedding technique. Tissue sections of 3–5 μm in thickness were obtained using a microtome (Leica 2135) and were then stained with haematoxylin and eosin as described by Suvarna et al. [20 ]. The tissue sections were finally examined via light microscopy, and lesions were photographed using an Olympus XC30 camera (Tokyo, Japan). Gram’s stain was carried out in tissue sections of urinary bladder to demonstrate Gram negative and Gram positive bacterial aggregations in the examined tissue [20 ].

Integrity of the tight junction was assessed using immunofluorescent staining of proteins zonula occludens 1 (ZO-1) and occludin. After the exposed cocultures were washed, fixed with ice-cold methanol for 10 min at −20 °C and permeabilized with 0.3% Triton-X100 for 10 min, the coculture was blocked with 1% BSA solution for 1 h and incubated successively with Alexa Fluor^® 488-conjugated mouse anti-human ZO1 antibody (1/100, ThermoFisher, Gent, Belgium) and Alexa Fluor^® 594-conjugated mouse anti-human Occludin antibody (1/100, ThermoFisher, Gent, Belgium) for 2 h in 0.5% BSA solution. The nuclei were counterstained with DAPI (1/500) for 10 min. Finally, the membranes were excised from the insert and mounted using ProLong^® Gold Antifade Mountant (ThermoFisher, Gent, Belgium). Images were taken with an Olympus BX61 microscope using the XC30 camera at ×40 magnification.

After 24 h, 48 h, 72 h, 96 h, 120 h and 144 h of incubation in darkness at 20 °C and 60% relative humidity in an Innova 4900 growth chamber (New Brunswick Scientific, Edison, NJ, USA) at 120 rpm, 30 μL of each sample was applied on a sterile slide, observed and photographed using an Olympus BX53 Upright Microscope equipped with an Olympus XC30 camera. The presented results are the percentage of germinated macroconidia and the average length of hyphae of 300 conidia from 3 independent experiments/replicates (100 spores per one repetition) observed in 10 different microscopic fields. The obtained results were calculated in relation to an appropriate control (water, RB).