Evos auto 2

To observe the amyloplasts in bitter pit cells, the pulp was soaked in a fixative (5% (v/v) formaldehyde, 5% (v/v) acetic acid, and 45% (v/v) ethanol) at 4°C for 48 h. The fixed pulp was stained with an I₂-KI solution (0.15% (w/v) I₂ and 0.45% (w/v) KI) for 5 min. Then, the fruit pulp was observed under a light microscope (EVOS Auto 2, Thermo Fisher Scientific, MA, United States) (Takahashi et al., 2003 (link)).

The PDA medium (20 ml) was poured into a 9-cm-diameter Petri dish. One mycelium disc (0.5 cm in diameter) of the fungus B. dothidea was inoculated in the center of the Petri dish. Then one disc of P. protegens was inoculated on each side 2 cm from the fungal disc (Pp). Sterilized water (50 μL) was used as the control (CK). All the Petri dishes were inverted and incubated at 28°C in the dark for 3 days. The diameters of the fungal colony were measured daily to evaluate the inhibition of P. protegens on the mycelial growth B. dothidea. After that, P. protegens-treated mycelia and the control mycelia were carefully picked from the Petri dishes and observed under a microscope (EVOS Auto2, Thermo Fisher Scientific, United States) with × 40 magnification. The experiments were repeated three times, and five replicates were included for each sample in each experiment.

A layer of cellophane was laid on a newly prepared PDA medium. One fungal mycelium disc (0.5 cm in diameter) was inoculated on the cellophane. S. plymuthica culture (50 μl) (OD₆₀₀ = 0.6) was added onto a small piece of filter paper (1 × 1 cm), 1 cm away from the mycelium disc, over the cellophane. The fungal mycelium disc without S. plymuthica was used as an untreated control. The Petri dishes were inverted and incubated at 28°C in the dark. Twenty-four hours later, the cellophane with new mycelium was taken out from the Petri dishes, cut into 1 cm × 1 cm squares, and was observed under a microscope (EVOS Auto2, Thermo Fisher Scientific, United States).

After a 4 hour Chandler loop rotation, blood was drawn out of the loops and the loops were flushed with 30 ml of PBS to remove excessive cells which were not firmly adhering to the grafts surfaces. BNC grafts were cut and embedded in Tissue-Tek O.C.T. compound (Sakura Finetek Europe B.V., Alphen aan den Rijn, NL) and stored at -80°C. After freezing, cryosectioning was performed using a Leica CM 1950 cryostat (Leica Biosystems, Nussloch, GER). Here, activated thrombocytes were detected using rabbit anti-human CD62P (Biorbyt #orb416329) as primary antibody and respective donkey anti-rabbit Alexa Fluro 488 (Jackson ImmunoResearch #711-547-003) as secondary antibody. Likewise, erythrocytes were detected using mouse anti-human CD235a (Biorbyt #orb248903) as primary antibody and respective donkey anti-mouse IgG Cy3 (Jackson ImmunoResearch #715-167-003) as secondary antibody. Upon acetone fixation, the graft slices were blocked with 3% standard donkey serum and then incubated with the primary antibodies at the dilution of 1:50 in 3% donkey serum overnight. After washing, the fluorescence dye conjugated secondary antibody at the dilution of 1:500 in 3% donkey serum was incubated on the slice in the dark for one hour. The slices were then counterstained with DAPI (Sigma D9542) to stain the nuclei before mounting and microscopy (EVOS Auto 2, ThermoFisher Scientific, Massachusetts, USA).

FDA (Fluorescein Diacetate, Thermo Fisher, USA) was dissolved in DMSO (Dimethyl Sulfoxide) to produce a 1 mg/ml stock solution. One microliter of stock solution was added to 99 μL of DMSO to prepare a working solution. Then, 99 μL of protoplast suspension was placed into a 0.2 ml centrifuge tube, and 1 μL of the FDA working solution was added. The staining was carried out for 5 min at 25 °C in the dark. Before observation, the stained sediments of protoplast suspensions were washed three times with basic solution by centrifugation at 1000 rpm. Then, the viability of the protoplasts was tested under a fluorescence microscope (EVOS Auto 2, Thermo Fisher, USA). We selected the light cube of GFP because the excitation wavelength of FDA is 490 nm^{60 (link)}.

Intracellular ROS production was determined by staining A549 cells with an oxidation-sensitive fluorescent probe 2,7-dichlorodihydrofluorescein diacetate (DCF-DA) according to the manufacturer’s protocol. Briefly, A549 cells were treated as indicated for 48 h. Subsequently, the medium was removed, and the cells were incubated with DFCH-DA (Beyotime Institute of Biotechnology, Haimen, China) at a final concentration of 10 μM at 37°C for 20 min. Afterwards, the cells were washed with buffer solution three times as per instructions. The fluorescent intensity was photographed and analyzed with a fluorescence microscope (EVOS^TM Auto 2, Invitrogen, WA, USA).