Lsm 510 meta

Nitric oxide, O₂^·− and cell viability were detected in rice root of control and treatments (SNP, AsIII and AsIII + SNP) on 4^th and 12^th day. Fluorescent probe 4-aminomethyl-2′, 7′ difluorofluorescein diacetate (DAF-FM DA, Calbiochem) was used to detect NO in root^{69 (link)}. Root was incubated in 10 µM DAF-FM DA (in 1x PBS, pH 7.2) for 30 minutes at 25 °C and washed thrice with PBS (for 5 minutes each) after staining^{69 (link)}. Superoxide radicals (O₂^·−) were also detected using 10 µM dihydroethidium (DHE, Calbiochem)^{70 (link)}. The microscopic observation was performed under a confocal microscope (LSM510 META, Carl Zeiss) with 10x Plan-Apochromat lenses using λex- 488 nm and λem- BP505–550 nm for NO detection and λex- 514 nm and λem- LP560 nm for superoxide radicals. Rice root cell viability was performed by incubating the root samples in the fluorescein diacetate (FDA, Sigma Aldrich, USA), propidium iodide (PI, Sigma-Aldrich, USA) for [2 mg/ml (FDA) in acetone was diluted by PBS drop by drop until the solution turned milky with 1 µg/ml PI (PBS) added to a final concentration] 5 minutes. The images were captured by using green (FDA; λex- 488 nm and λem- BP505–550 nm) and red channels (PI; λex- 543 nm and λem- LP560 nm) of the confocal microscope (LSM510 META, Carl Zeiss).

Incorporation of 5‐ethynyl‐2′‐deoxyuridine (EdU) into nuclear DNA was detected using iClick™ EdU Alexa Fluor 488 Imaging Kit (Catalogue number A003) according to the manufacturer’s instructions (Applied BioProbes), as previously described (34 ). Incorporation of this thymidine analog in a punctate pattern, in keratinocytes cultured without growth factors, measures unscheduled DNA synthesis rather than DNA replication (38 (link)). Visualization of nuclear EdU‐incorporation was carried out by confocal laser microscopy (LSM 510 Meta; Zeiss, Oberkochen Germany). Densitometry was performed using Image J software (National Institute of Health, Maryland). Stained cells were manually counted from the images taken by Zeiss LSM 510 Meta confocal microscope. Fluorescence intensity was measured in cells that were above the detection threshold and considered "UDS positive."

NIH3T3 cells were stained with MitoTracker Red CMXROS (Molecular Probes, Eugene, OR, USA) for 30 min at 37 °C, washed with PBS, fixed with methanol and acetone, washed, and blocked with normal goat serum. The cells were subsequently stained at 4 °C overnight with a monoclonal anti-α-tubulin antibody (Sigma-Aldrich) and 4′,6-diamidino-2-phenylindole (DAPI) to stain nuclei. The mean fluorescence intensity (MFI) was measured using LSM 510 Meta software (Carl Zeiss, Oberkochen, Germany). For analyses of T helper cell populations, spleen tissue sections (7 μm thick) were fixed and stained with Alexa 488–conjugated anti-CD4, PE-conjugated anti-IL-17, APC-conjugated anti-CD25, and PE-conjugated anti-Foxp3 antibodies (eBioscience). The stained sections were visualized by confocal microscopy (LSM 510 Meta; Carl Zeiss).

Cells were cultured onto 12 mm glass coverslips coated with FBS and fixed with paraformaldehyde (PFA) for 15 minutes at room temperature (RT); PFA-fixed cells were then permeabilized in 0.1% saponin in PBS for 10 minutes before subsequent incubation in 50 mM NH₄Cl in PBS for 10 minutes and in blocking solution (composed of 0.2% BSA and 0.2% fish skin gelatin in PBS) for 20 minutes at RT. A 30-minutes incubation in blocking solution containing primary antibodies was followed by 4×5 minutes PBS washes before incubation in blocking solution containing fluorescently tagged secondary antibodies for 20 minutes. After further 4×5 minutes PBS washes, the cells were incubated with DAPI for 1 minute just before rinsing in water and then mounted in Mowiol. Image acquisition of cells, indirectly labeled with Alexa fluorophores (Invitrogen, Life Technologies, Monza, Monza Brianza, Italy), was performed at RT in Acqua-Poly Mount medium (Polysciences, Warrington, PA, USA) using a confocal microscope with photomultiplier tube detectors (LSM 510 Meta; Carl Zeiss, Oberkochen, Germany) equipped with a 63x oil immersion objective (Carl Zeiss Inc., Oberkochen, Germany). The data were captured using the LSM 510 Meta software (Carl Zeiss, Oberkochen, Germany) and unprocessed images were assembled using Photoshop (CS3; Adobe).

PCR-generated Sal1-Nco1 and Hind III- BamH1 fragments containing the open reading frame of GmZFP3, respectively, were subcloned in-frame upstream of the GFP gene in the pJIT166GFP plasmid. All constructs were validated by sequencing. The primer sequences are listed in Table 1.
Arabidopsis leaf protoplasts were isolated according to Yoo et al. (2007 (link)). The two resulting fusion constructs or empty control vector (p35S::GFP) were introduced into Arabidopsis protoplasts by the PEG4000-mediated method (Abel and Theologis, 1994 (link)). After incubation of transformed Arabidopsis protoplasts for 18−24 h at room temperature, GFP signal was detected by confocal fluorescence microscopy (Zeiss, LSM510 Meta, Carl Zeiss AG).
To further validate GmZFP3 localization, full-length GmZFP3 CDS lacking a stop codon (primers used are listed in Table 1) were amplified and cloned into the pMDC83 destination vector using the Gateway™ method. The in-frame GFP fusion constructs were transformed into the Agrobacterium tumefaciens GV3101 strain by electroporation and injected into the tobacco leaf. GFP signal was examined by confocal fluorescence microscopy (Zeiss, LSM510 Meta, Carl Zeiss AG). Nuclei were stained with DAPI (4′,6-diamidino-2-phenylindole).