Tcs sp8 sr

Cellular imaging experiments were carried out as described previously to verify the utility of cel-p for imaging of potential cellular targets [23 (link)]. To track the cellular locations of cel-p, living primary neurons were incubated with cel-p 0.8 μM for 0–6 h. The cells were fixed with 4% paraformaldehyde solution for 10 min and 0.2% Triton X-100 permeated for 15 min. Click chemistry reaction was carried out (regents and concentrations as described above) for 2 h and washed thrice to remove excessive agents. The cells were stained with Hoechst for 10 min. DMSO-treated samples were used as control concurrently. Imaging was done with confocal fluorescence microscopy (Leica TCS SP8 SR, Germany).
For colocalization experiments, living primary neurons were incubated with DMSO, 0.8 μM cel-p in the presence or absence of competitor (cel, 8 ×) for 4 h. The cells were fixed, permeated, carried out with click chemistry reaction, and washed as described above. The cells were incubated overnight at 4 °C with anti-HMGB1 antibody and then with secondary fluorescence antibody (goat anti-rabbit, 1:500, Abcam) for 2 h at r.t.. The images were obtained with confocal fluorescence microscopy after staining with Hoechst for 10 min.

Full-length sequences of the four 3′ UTRs of AhACCA1 were amplified using primers (GFP-1-F-Sal I, GFP-1-R-Hind III, GFP-2-R-Hind III, GFP-3-R-Hind III, GFP-4-F-Sal I, and GFP-4-R-Hind III, Table S6) containing the sites for the restriction enzymes Sal I and Hind III and cloned into the pCAMBIA1300-35S-EGFP vector. The E9 terminator of EGFP was replaced to verify the functionality of the 3’ UTRs. The resulting clones were named EGFP-UTR1, EGFP-UTR2, EGFP-UTR3, and EGFP-UTR4 and were individually transformed into Agrobacterium (Agrobacterium tumefaciens) strain GV3101 according to Gui et al. [51 (link)]. The appropriate cell density of each bacterial culture was infiltrated into Nicotiana benthamiana epidermal cells. The plants were placed in the dark for 1 day and then in dysphotic conditions (illumination intensity: ~50 lx) for 1–2 days before peeling off the leaf abaxial epidermis for observation using a confocal microscope (Leica TCS-SP8 SR, German). An Agrobacterium suspension harboring the pCAMBIA1302-35 S-EGFP empty vector was used as the control.

The measurements of the ROS level in the guard cells were performed with H₂DCFDA. The epidermal strips were transferred to 10 mL of loading buffer (10 mM Tris, 50 mM KCl, pH 6.5) and treated with 50 µM H₂DCFDA in the dark for 30–60 min. Afterward, the epidermal strips were washed with loading buffer to remove the excess dye. The fluorescence was determined using confocal microscopy (TCS SP8 SR; Leica) with an excitation wavelength of 460–500 nm.

Cells affixed to slides were incubated in a fresh medium containing 200 μM TMZ/DMSO for 48 h. After three washes with PBS, cells were fixed with 4% paraformaldehyde and then left in 0.2% Triton for 10 min to disrupt the cell membrane. 200 μL of blocking solution was added to each well and the cells were treated for 1 h at room temperature. Anti-GINS2 antibody (Proteintech, #16247-1-AP, 1:200) and anti-γH2AX antibody (Abmart, #M63324S, 1:200) was used to culture the cells overnight at 4 °C. Goat Anti-Rabbit IgG (Dylight 594, Abbkine, A23420, 1:200) and Goat Anti-Mouse IgG (DyLight 488, Abbkine, A23210, 1:200) were used to conjugate the corresponding primary antibodies for 1 h. After washing again, the nuclei were stained with DAPI for 30 min and sealed with neutral gum. Fluorescence and colocalization were observed with laser scanning confocal microscopy (Leica, TCS SP8 SR).

The cDNA sequence of ClDMP3 was amplified using gene-specific primers (Table S3) and ligated into the pGREEN vector to fuse with a green fluorescent protein (GFP) gene. The fusion protein ClDMP3-GFP was driven by a 35S promoter. AtCBL-RFP was also driven by a 35S promoter and was used as the membrane maker [55 (link)]. The two constructs were transiently co-transformed into tobacco (Nicotiana benthamiana) leaves through infiltration with Agrobacterium strain GV3101 (containing helper plasmid pSoup). After 48 hours, a confocal laser scanning microscope (Leica TCS-SP8 SR, Germany) was used to observe fluorescence.

The pGREEN vector fused with green fluorescent protein (GFP) was used to determine the subcellular localization of the ClCOMT1 protein. The cDNA sequence of ClCOMT1 was PCR-amplified with primers containing EcoRV and XhoI restriction sites. The amplified products were gel-purified and ligated into the pGREEN vector. Then, the recombinant plasmid pGREEN-ClCOMT1-GFP was transiently transformed into watermelon leaf protoplasts. Protoplast extraction and transfection were performed as described previously using 20-day-old watermelon leaves^{43 (link)}. Fluorescence was observed using a confocal laser scanning microscope (Leica TCS-SP8 SR, Germany).

The AGAP2-ASA and miR-9-5p localization in cells was determined using the FISH technique. RiboTM lncRNA FISH Probe Mix (Red) (Ruibo Biotechnology, China) was used according to the manufacturer's instructions [47 (link)]. The specific method was as follows: coverslips were placed in a 6-well culture plate, and the test cells (1 × 105 cells/well) were seeded into the wells and incubated for 1 day to reach a cell fusion rate of around 80%. The coverslips were removed, washed with PBS, and fixed with 1 mL of 4% paraformaldehyde at room temperature. After treatment with proteinase K (2 μg/mL), glycine, and acetylation reagent, 250 μL of pre-hybridization solution was added and incubated at 42 °C for 1 h. The pre-hybridization solution was removed, and 250 μL of hybridization solution containing the probes (300 ng/mL) was added and hybridized overnight at 42 °C. After washing three times with PBST, the nuclei were stained with DAPI diluted in PBST (1:800) and incubated for 5 min in a 24-well culture plate. The samples were then washed three times with PBST for 3 min each. Finally, the coverslips were mounted with an anti-fluorescence quenching agent and observed and photographed using a laser confocal microscope (Leica, TCS-SP8 SR, Germany) with five fields of view selected for analysis.