Dp74 camera

For histopathological examination, cardiac tissues were collected from all studied groups and dissected and fixed in 4% formaldehyde solution. The heart samples were embedded in paraffin, and five micron-thick sections were sliced. Standard histological methods (xylol) were used in order to remove paraffin, and the samples were passed through a gradual alcohol series and hydrated. Trichrome staining was used to demonstrate the general histological structure; it is a three-color staining process, which differentially stains the nucleus, muscle tissue, and collagen. It was performed by using Trichrome Stain Kit (Abcam, Cambridge, UK). The sections were studied under Olympus CX-31 microscope and photomicrographs were taken using Olympus DP74 camera (Olympus Corporation, Tokyo, Japan) at 4× and 40× magnification. The remaining portion of the heart was stored at −80 °C for lipid peroxidation assay and Western blot analysis.

Seminal fluid was collected from the cauda epididymides of male mice at 8 months of age and the weight was measured. The sperm were incubated with the medium (FERTIUP Kyudo Co. Ltd. Saga, Japan) containing Hoechst 33348 (1.5μg/ml, Sigma Aldrich) for 1 hour. After incubation, the medium containing sperm was diluted with PBS and the number of sperm was counted. For analysis of sperm morphology, the sperm was observed by fluorescence microscope (All-in-one Fluorescence Microscope BX-X710, KEYENCE, Osaka, Japan). Further, the diluted medium containing sperm was smeared to slide glasses and the smears were stained with Wright-Giemsa and analyzed with a Zeiss Axioskop 2 plus microscope (Carl Zeiss, Tokyo, Japan) with a Olympus DP74 camera (Olympus Co., Tokyo, Japan).

Seeds of tomato (S. lycopersicum ‘Ohgata‐Fukuju’) plants were sterilized with 70% ethanol solution for 20 s and 1% sodium hypochlorite solution for 20 min (Inoue et al., 2023 (link)). The seeds were sown on 1% agar in Petri dishes at 30°C under dark and axenic conditions. Four days after sowing, seedlings with approximately 20‐mm‐long roots were placed on agar plates, and 200 μL of OE1‐1 suspension at 10⁸ cfu/mL was added to the centre of the plate, 10 mm away from the tomato roots. The tomato seedlings were then incubated at 30°C under dark and axenic conditions. Root pieces were excised 4 mm from the tip, fixed in half‐strength Karnovsky's fixative overnight at 4°C, and then post‐fixed in 1% osmium tetroxide for 1 h at room temperature (Karnovsky, 1965 ). The specimens were dehydrated in an ethanol series (three times at 30%, 50%, 70%, 80%, 90%, and 100%) for 10 min each at 25°C and then embedded in Quetol‐812 resin (Nisshin EM). Serial semi‐thin sections (800 nm thick) were cut with a diamond knife, mounted on glass slides, stained with toluidine blue, and observed under a BX53 optical microscope equipped with a DP74 camera (Olympus).

Images were taken at room temperature using an Olympus CKX41 microscope and Olympus DP74 camera. Cellsens software (Olympus, Tokyo, Japan) was used to acquire images on the microscope.

The possible morphological changes in HGF cells after exposure to the extraction medium were assessed by comparing photographs between control cells (un-stimulated cells) and treated cells. All pictures were taken at magnification 10×, using an Olympus IX73 inverted microscope documented with DP74 camera (Olympus, Tokyo, Japan).