Bx51 microscope

The study parameters examined included nuclear area (NA), cytoplasmic area (CA), ratio of nuclear area to cytoplasmic area (N/C), nuclear roundness factor (NRF), nuclear length (NL), nuclear width (NW), and nuclear perimeter (NP). The cytomorphometric examinations were carried out by two of the authors (Sibel Bektas, Zehra Safi Oz) blinded to the study and control groups. The cytological analysis was performed using digital photographs obtained from the slides using an Olympus BX 51 Microscope (Tokyo, Japan) and a mounted Leica DFC-280 digital camera (Germany). Measurements of the nuclear and cytoplasmic areas were calculated on digital images using a line encircling the nuclear and cytoplasmic boundaries of the cells (Figure 1).

GFP fluorescence was observed with Olympus BX-51 microscope and Leica DFC310-FX CCD imager, with a fixed exposure condition for each series of imaging. Sperm motility was filmed at 200 frames per second. Flagella curvatures and swimming velocity were measured using image analysis software BohBoh (Bohboh Soft, Tokyo, Japan).

Karyotyping was carried out as described previously (Nehra et al., 2022 (link)). Briefly, hiPSCs were blocked at metaphase by exposure to 0.1 μg/ml Colcemid solution for 3 hrs. (Gibco). Karyotyping of isogenic euploid and Down syndrome hiPSCs was performed using G-banding with a resolution of 350–400, using Olympus BX51 microscope and Cytovision software (Leica).

Fixed tissues were embedded in paraffin. From paraffin blocks, 5 μm thick sections were made using a Leica SM 2000 R microtome. The sections were stained with hematoxylin and eosin (H&E) and Masson’s trichrome, and immunohistochemistry using the antibody against α-smooth muscle actin and Kv11.1 channel (Alomone Labs, Jerusalem, Israel) was carried out. Histological specimens were examined using a Leica BX 51 microscope, a Leica MC 190 digital camera, and the Leica LAS software at a magnification of x200. Five photographs were taken for each placenta sample and representative images were selected randomly. Morphometric investigations included the assessment of placental arterial wall thickness and the placental arterial lumen index (the ratio of the internal vessel area to the external vessel area) using the ImageJ software. All the sample preparations were randomized, and the data analyses were performed in a blinded manner.

For fluorescent microscopy, young adult worms were immobilized by using 0.1 M levamisole or 10 mM NaN₃ in M9 buffer. Images for quantitative analysis were taken by using an Olympus BX51 microscope (Shinjuku, Tokyo, Japan), Leica DMI6000B microscope (Leica Camera AG, Wetzlar, Germany), or Zeiss AxioImager Z1. To observe AAK-2::GFP expression upon oxidative stress, age-synchronized L4 larvae were transferred to TBHP assay plates containing 1, 2, or 4 mM TBHP; NGM or RNAi plates without TBHP were used as control. Images of whole animals were taken after 1, 5, or 24 h incubation, and fluorescence intensity was measured by using ImageJ software. At least 15 worms were analyzed in all conditions, and the experiments were repeated at least 3 times. To study GFP::LGG-1 puncta upon oxidative stress, young adult age-synchronized worms were incubated on TBHP assay plates (1 mM TBHP) for 3 h. Epifluorescence images of the whole animals and the first 2 intestinal cells behind the pharynx were taken and analyzed by using ImageJ software; the GFP::LGG-1 positive area were measured relative to the area of the cell on each image. Up to 36 worms were analyzed in all conditions, and the experiments were repeated 2 times. All experiments were conducted at 20°C. Statistical significance was determined by using independent 2-sample Student’s t tests.

U-2OS cells (5×10³ cells/ml) were seeded onto the different surfaces of the SAMs. Following a 3-, 6- and 9-h culture, cell adhesion analyses were performed under an inverted microscope (HBOSO/AC Hg lamp, Oberkochen, Germany; BX-51 microscope, Leica, Germany). Further adhesion analysis of the cells cultured for 6 h was performed by scanning electron microscopy (SEM; Hitachi, H50, Hitachi, Ltd., Tokyo, Japan).
The U-2OS cells attached to the surface of the SAMs could be visualized clearly under the inverted phase contrast microscope, so three different time points were selected to for investigation. The cell morphology was examined, and the attached cell numbers were counted in five random fields.
The morphology of the cells on the sample surfaces was further examined by SEM. Following cultivation for 6 h, the samples were removed from the culture plates and fixed with 3% glutaraldehyde (Sigma-Aldrich, St. Louis, MO, USA) for 6 h. Following this, they were washed three times with PBS (10 min each time) and dehydrated sequentially in a series of ethanol (50, 70, 95 and 100%; each concentration twice for 10 min each time) and air dried in a fume hood. Following sputter-coating with gold, samples were examined by SEM, and the adhesion and spreading of the cells in the different groups with modified substrates were observed by SEM (Hitachi, H50).