Lsm 980 laser scanning microscope

Arabidopsis Col-0 seeds harboring pOpON-AtCDKA;1(DN)-GFP construct were sown on B5 medium supplemented with 2% (w/v) sucrose and containing 1.5% (w/v) agar, stratified at 4°C for 3 days in the dark, and then exposed to white light for 24 hours. After germination, plates were placed vertically at 22°C in the dark for 2 days, followed by treatment with 30 μM DEX, or the equivalent volume of DMSO for mock treatment, and incubated for 1 day for induction. After confirmation of induction by DEX, as indicated by GFP fluorescence, seedlings were transferred to B5 medium without sucrose and containing 1.5% (w/v) agar in square plates and then illuminated with unilateral BL. Phototropic response was examined by measuring the hypocotyl angle after 2 days of irradiation using ImageJ. To observe the induction by DEX in the hypocotyl, a ZEISS LSM 980 laser scanning microscope equipped with a 40×/water objective lens was used to excite chlorophyll autofluorescence at 639 nm. Autofluorescence was captured between 641 and 694 nm. For the detection of GFP, the excitation wavelength was 488 nm, and the emission was collected between 490 and 596 nm.

HepaRG and HepG2 cells were grown on cover slides for indirect immunofluorescence. At different times posttransfection or infection, the samples were fixed by incubation in 4% paraformaldehyde (PFA) for 10 min at room temperature. For HBV-infected HepG2 cells, an additional CSK washout was performed in indicated samples as recently described (42 (link)). Prior to blocking in Tris-buffered saline–BG (TBS-BG; BG is 5% [wt/vol] bovine serum albumin [BSA] and 5% [wt/vol] glycine) buffer for 30 min at room temperature, the samples were permeabilized in 0.5% Triton X-100 for 5 min. The cover slides were incubated with primary antibody diluted in phosphate-buffered saline (PBS) overnight at 4°C. Samples were washed three times with TBS-BG and incubated in the corresponding Alexa Fluor 488 (Invitrogen)- or Alexa Fluor 647 (Dianova)-coupled secondary antibodies. Prior to mounting in Mowiol, the slides were washed three times in TBS-BG. Samples were analyzed using a confocal laser-scanning microscope (Nikon TiE) and the Volocity software, a Zeiss LSM 980 laser scanning microscope and the Zeiss Zen 3 software, or a Zeiss Axio Observer Z.1 microscope and the Axiovision 4.8 software. To quantify colocalization, Spearman correlation ranks of the respective fluorescence channels were determined using Fiji (76 (link)) and the Pearson-Spearman correlation colocalization plugin (77 (link)).

Propidium iodide (PI) staining was performed as previously described [10 (link)]. For confocal microscopy, fluorescence in roots was detected using a Zeiss LSM980 laser scanning microscope (ZEISS, Oberkochen, Germany). The PI signal was visualized using 561 nm as the excitation wavelength and 591 to 635 nm as the emission wavelengths. GFP fluorescence was detected using 488 nm as the excitation wavelength and 510 to 530 nm as the emission wavelengths. YFP fluorescence was detected using 514 nm as the excitation wavelength and 530 to 600 nm as the emission wavelengths. Images and fluorescence intensities were processed using Zen 2012.