Tcs sp5 2

To determine the abundance and positioning of CDR1as in tissues and cells, RNA FISH assays were performed as described by Cui et al [31 (link)] Briefly, the DNA probe targeting the end-to-head junction of CDR1as was labeled with FITC. After fixing in a 4% (wt/vol) paraformaldehyde solution, samples were rinsed in 1 × PBS, permeabilized in 1 × PBS with 0.5% (vol/vol) Triton X-100 (10 min), washed in 1 × PBS with 0.1% (vol/vol) Tween-20 (1 min). Probes were mixed with pre-made hybridization buffer, and then samples were incubated in hybridization buffer at 37 °C overnight. After washed with hybridization buffer at 37 °C for 15 min and quickly rinsed at room temperature three times, cells were stained in DAPI stain solution. Finally, images were taken under immunofluorescence microscope (Leica, TCS SP5II).
For protein IF assays, cells were cultured on glass slides in petri dishes. When nearly confluent, cells were fixed with 4% paraformaldehyde for 10 min, permeabilized in 0.5% Trion-X-100 for 15 min and blocked in 3% BSA for 30 min at room temperature. Then cells were incubated with the primary antibody against p53 or Phospho-Histone H2A.X overnight at 4 °C, followed by incubation with the secondary antibody conjugated with FITC for 45 min at 37 °C. Immediately after DAPI was added, images were taken with immunofluorescence microscope (Leica, TCS SP5II).

Scanning was conducted on a confocal laser-scanning microscope (Leica TCS SP5 II). For optimal light transmission, tissues were cleared in 98% methyl salicylate (Merck, Darmstadt, Germany; Cat-no. W274518). After dehydration in ethanol, brains that were previously fixed in ZnFA (n = 7) as well as in half-strength Karnovsky’s solution (n = 6) were transferred into custom-made scan chambers filled with pure methyl salicylate before confocal laser-scanning. Scanning was performed with an inverted Leica TCS SP5II (Leica, Wetzlar, Germany) using a DPSS-laser with an excitation wavelength of 561 nm and a speed of 400 Hz. For detection of fluorescence (emitted by glutaraldehyde-enhanced autofluorescence as well as by Cy3-conjugates of the secondary antibody), a 10× objective with a numerical aperture of 0.4 was used resulting in stacked images of 1,024 × 1,024 pixels with a pixel size of about 0.8 μm. The confocal microscope operated with a pinhole size of 53 μm in diameter and in steps of 1.33 μm (system-optimized to one airy unit and refractive correction for aqueous immersion media).

For subcellular localization experiments, the GV3101 strains harboring pHB-AaTCP14-YFP or pHB-YFP were transformed into 5-week-old N. benthamiana leaves. YFP signals were analyzed 60 to 72 hours after infiltration by confocal laser microscopy (Leica TCS SP5-II). Nuclei were stained with DAPI (Sigma-Aldrich, USA). Three biological repeats were performed to verify these results.
For two- and three-protein colocalization assays, the full-length cDNAs of AaORA, AaTCP14, and AaJAZ8 were ligated into the pHB-CFP and pHB-YFP vectors to obtain AaORA-CFP, AaTCP14-CFP, AaJAZ8-CFP, and AaJAZ8-YFP. GV3101 strains harboring AaTCP14-CFP and AaJAZ8-YFP, AaORA-CFP and AaJAZ8-YFP, or AaTCP14-nYFP, AaORA-cYFP, and AaJAZ8-CFP were cotransformed into 5-week-old N. benthamiana leaves. Meanwhile, GV3101 strains harboring AaORA-CFP and YFP, AaTCP14-CFP and YFP, CFP and AaJAZ8-YFP, or AaTCP14-nYFP, AaORA-cYFP, and CFP were also transformed into 5-week-old N. benthamiana leaves as negative controls. After incubation at 23°C for 60 to 72 hours, YFP and CFP signals were observed by confocal laser microscopy (Leica TCS SP5-II). Three biological repeats were performed to verify these results. The primers are listed in table S1.

For second harmonic generation (SHG), imaging samples were investigated by confocal microscopy (TCS SP5 II Leica) combined with MPM where the NIR femtosecond laser beam was derived from a tunable compact mode-locked titanium: sapphire laser (Chamaleon Compact OPO-Vis, Coherent). Two-photon excited fluorescence was used to induce SHG and obtain high-resolution images of unstained collagen structures. The samples were observed by using λ_ex = 840 nm (two photons) and λ_em = 415–425 nm. The SHG images were acquired with a resolution of 12 bit, 1024 × 1024 pixel by using a 25× water immersion objective (HCX IRAPO L 25.0X0.95 Water, n.a. 0.95).