U cmad3

Cell death was assessed using cell staining with acridine orange (AO) and ethidium bromide (EtBr), as described previously [24 (link)]. MCF-7 and MDA-MB-231 cells (0.3 × 10⁶ cell/2.0 mL medium/well) were grown in 6-well plates for ~36 h and then treated with vit-D3 (50, 100, and 200 µM) or vehicle 0.5% DMSO (controls) for 48 h. Following this, cells were trypsinized to obtain a single-cell suspension. Trypsin was neutralized in a complete medium, and a 20 µL cell suspension was transferred to a different 1.5 mL microcentrifuge tube and incubated with EtBr (100 µg/mL) and acridine orange (100 µg/mL) mixture for 5.0 min. The stained cells were placed on slides and photographed using a fluorescence microscope (Olympus U-CMAD3) with fluorescein isothiocyanate (FITC) and tetramethylrhodamine isothiocyanate (TRITC) probes. Merged images with green (live) and orange (dead) cells were analyzed.

Several transverse 10 µm sections were obtained from muscle samples mounted in OTC. Sections were collected at –20 to 22°C on the surface of a polarized glass slide. Cryosections were fixed with methanol in ice for 15 min, washed three times (5 min each) in PBS (NaCl 136 mm, KCl 2 mm, Na₂HPO₄ 6 mm, KH₂PO₄ 1 mm) at room temperature (RT) and incubated in 1% Triton X‐100 in PBS for 30 min (RT). Cryosections were then incubated with blocking reagents (4% BSA in 1% Triton X‐100 in PBS + 5% goat serum) for 30 min (RT), raised with PBS (three times for 5 min each) and probed with anti‐CD31 (dilution 1:100; Abcam, Cambridge, UK) overnight at 4°C. After three washes (5 min each) in PBS, cryosections were incubated with Alexa‐Fluor 488 anti‐mouse (dilution 1:200; Abcam) for 60 min at room temperature. Finally, the samples were probed with anti‐dystrophin (dilution 1:500 dilution) for 60 min at room temperature and then with Alexa‐Fluor 488 anti‐rabbit (dilution 1:200; Abcam) for 60 min at room temperature. Fluorescence intensity was visualized by microscopy (U‐CMAD3; Olympus). In total, 125–150 fibres were measured in each sample. Capillary density was defined as total number of capillaries per CSA of the associated muscle fibres (Hepple et al., 2000 (link); Hoppeler et al., 1981 (link); Mathieu‐Costello et al., 1991 (link), 1988 (link), 1992 (link)).

Semen samples were collected at the clinic by masturbation into a sterile plastic container. The samples were liquefied for 30 min in 37 °C before analysis. Macroscopic examination of semen was performed according to the 5th edition of WHO laboratory manual for the examination and processing of human semen [17 ]. Microscopic measurements of the sperm count, concentration, motility and morphology were determined with the use of computer-aided semen analysis (CASA). The basic components of the system were a bright field microscope (Olympus CX41, Tokyo, Japan), a digital camera to capture images (Olympus U-CMAD3), and a computer with software installed (SCA^®Microptic S.L., Barcelona, Spain). The WHO [17 ] cut-off points were used to evaluate abnormal values of semen quality parameters (Table 1). All analyses were performed by an experienced technician.

Light microscopic technique to examine the effects of the fungal extract on the initial and pre-formed biofilms was performed according to Taufiq and Darah (14 (link)). The biofilm formation was examined under the light microscope attached with a digital camera (Olympus U-CMAD3).

Six individual flowers from six different plants per species were fixed in a formalin acetic acid–alcohol solution (37% formaldehyde, glacial acetic acid, 95% ethanol, and deionized water in a 10:5:50:35 mixture) for microscopic analysis. The samples (floral stem, petal, lip, dorsal sepal, and synsepal) were cleaned with water before anatomical analysis. Thin transverse cross-sections (20–30 μm) of the samples were made with a Microtome Cryostat (CM3050S; Leica, Germany); the sections were stained with 1% fuchsine for 4–5 s, mounted on glass microscope slides, examined, and photographed with a light microscope (U-CMAD3; Olympus Inc., Tokyo, Japan). Epidermal thickness; exodermal thickness; endodermal thickness of the floral stem; upper epidermal thickness; lower epidermal thickness; mesophyll thickness of the petals, lips, dorsal sepals, and synsepals; and the whole petal, lip, dorsal sepal, and synsepal thickness were determined with the ImageJ program.

Muscle fibre CSA was determined from several transverse sections (10 µm thick) obtained from muscle samples and probed with anti‐dystrophin antibody. Fluorescence images were visualized by microscopy (U‐CMAD3; Olympus, Tokyo, Japan). Fibre CSA was measured with ImageJ (NIH, Bethesda, MD, USA) and expressed in square micrometres. In total, 125–150 fibres per sample were measured.

The cells were plated in six-well plates and incubated for 24 h. Concentrations of PZH were then added to each well of the three experimental groups, and then incubated for 24 h together with the negative control group. Next, the cells were washed three times with PBS and stained with Hoechst 33258 (1 mg/l) for 15 min at 37°C. Images of the Hoechst 33258 fluorescence were then captured using inverted fluorescence microscopy (U-CMAD3; Olympus, Tokyo, Japan), prior to washing the cells three times with PBS. The percentage of positively-stained cells was calculated according to the images.