U tv0.5xc 3

To elucidate the expression pattern of GmPAP4 in soybean nodules, we cloned 2000 bp promoter sequences of GmPAP4 upstream of the translation start codon ATG, fused with β-glucuronidase (GUS) reporter gene (pGmPAP4:GUS). The resulting construct was introduced into the soybean hairy roots by Agrobacterium rhizogenes K599 and the transgenic hairy roots were then inoculated with rhizobia Bradyrhizobium diazoefficiens USDA110 for nodule development. After 4 weeks of rhizobia inoculation, transgenic hairy roots and nodules were stained as described previously and captured with a light microscope (Olympus U-TV0.5XC-3, Tokyo, Japan) [14 (link)]. For GUS activity, total nodule proteins were extracted and incubated in a mixture containing 10 mM 4-methylumbelliferyl β-D-glucuronide (MUG; Sigma Chemical Co., St.Louis, MO, USA) for 1 h at 37 °C. The fluorescence product of 4-methylumbelliferone (4-MU) was monitored using a VersaFluor™fluorometer (Bio-Rad, Hercules, CA, USA) with excitation at 365 nm and emission at 455 nm.

Corneas were flattened with four cuts at 12, 3, 6 and 9 o'clock positions, into four quadrants. The corneas were mounted with mounting media (Immu-Mount; Thermo Fisher Scientific, Inc. Kalamazoo, MI, USA) on slides, and photographs were taken under an Olympus Microscope (BX43; Olympus, Tokyo, Japan) attached to a digital camera (U-TV0.5XC-3; Olympus). The digital images of flat-mounted corneas were analyzed using the Olympus CellSens Standard 1.17 software (Olympus). The total area of the cornea was encircled by drawing a freehand region, including the innermost vessels of limbal arcade. The new vessel sprouts were connected using a freehand selection and then calculated. The total corneal area and the avascular area were quantified with the assistance of software. The percentages of NV area in the total cornea were calculated.

Optical microstructure (OM) analysis was done using OLYMPUS U-TV0.5XC-3 (BX53M). Using this reinforcement distribution and different phase formations, modifications that occurred in the heat-treated composites were identified. The hardness test was performed using a Micro Vickers Hardness tester, Model-MMTX7A, as per ASTM E384 standards [34 (link)]. During this test, the load was set at 200 gmf with a dwell time of 15 s. Five readings were taken for each sample, and the average hardness value was used in the graphs plotted along with the standard deviation. Hardness tests were carried out for as-cast (AC) samples and samples subjected to SSHT and MSHT, followed by artificial aging at 100 and 200 °C for different time intervals (0–16 h) until peak hardness was attained. A tensile test was performed only on AC and peak-aged (highest VHN value) samples of both the composites using an Electronic Tensometer, Model-PC-2000/605/06, as per ASTM E8 standards [33 (link)]. The load was maintained at 20 kN with a break test mode and speed of 1 mm/min. Fracture analysis for the broken tensile samples of both AC and peak-aged (highest VHN value) samples of both composites was performed using SEM, and the mode of fracture experienced by them was illustrated.

To investigate the potential formation of yolk sac vessels from stem cells, an in vitro test was performed using the BD Matrigel™ substrate (BD Biosciences, USA). As a positive control, bovine umbilical vein endothelial cells (BUVECs) cultured in vitro under the same conditions as those of bYS-MSCs. The Matrigel (BD Biosciences) was prepared according to the manufacturer's instructions.
The bYS-MSCs were plated at a concentration of 1 × 10⁵ cells in EGM-2 medium (Gibco-BRL, USA) with 10% FCS on the Matrigel substrate (Covas et al., 2008). Samples were viewed under phase-contrast microscopy (Olympus IX71), and pictures were recorded using a digital camera (Olympus U-TV0.5XC-3) at 0 hour, 10 hours, 24 hours, 5 days, 10 days, and 15 days. The area was quantified using the ImageJ software (NIH Image-BioLab). We did the comparative analysis of the umbilical cord and bYS-MSCs.

For microscopy analysis, whole buds and samples of bud scales, stipules, and young leaves were vacuum infiltrated with Karnovsky’s fixative (paraformaldehyde 4% and glutaraldehyde 5% in phosphate buffer 0.1 M, pH 7.2; modified from [62 ]) for 5 min and left to set for 24 h in the same solution. Soon after, they were dehydrated in an increasing ethanol series (10–98%) and embedded in (2-hidroxiethyl)-methacrylate (Historesin embedding kit, Leica, Heidelberg, Germany). Transverse and longitudinal sections of the entire apex and fragments of stipules, bud scales, and young leaves were obtained using a rotary microtome (Hyrax M40, Carl Zeiss Mikroskopie, Jena, Germany). The 5–6 µm thick sections were mounted on glass slides and stained with toluidine blue (0.5% in acetate buffer 0.1 M, pH 4.7; modified from [63 (link)]). Analysis and image capture were performed using a light microscope (CX41RF, Olympus Scientific Solutions, Waltham, MA, USA) coupled to a digital camera (U-TV0.5XC-3, Olympus Scientific Solutions, Waltham, MA, USA) and a computer with an imaging software (LCmicro, Olympus Soft Imaging Solutions, Waltham, MA, USA).