Ultraview vox

For clonal analysis, Tg(MAZe,Rx2:GFPcaax,UAS:Kaede) embryos were heat shocked either at 10 hpf or 54 hpf for 5-20 min at 37°C, and allowed to recover at 28.5°C. At 72 hpf, the embryos were screened on a Nikon Eclipse TE2000 upright fluorescent microscope with a 40× water-immersion objective for Rx2-positive retinas with Kaede-expressing cells. The selected embryos were then embedded in 3% methylcellulose and transferred to a spinning-disc microscope (Perkin Elmer UltraVIEW VoX) equipped for photoconversion coupled with an Olympus 40× water immersion objective (NA 1.3), where photoconversion was performed on green Kaede-expressing cells. For short-term analysis, at 3 dpf or 5 dpf, single cells in the CMZ region (defined by Rx2 membrane marker) were randomly targeted by applying a 5 s train of 405 nm laser pulses until the cell turned red. For long-term clonal analysis, the same photoconverting procedure was applied initially at 5 dpf but on all the cells in a connected Kaede cell patch in the CMZ region. The photoconversion was then repeated on the converted cells that still remain in the CMZ at subsequent time points, such as 6, 8, 12, and 16 dpf or until the death of the fish.

The CmbHLH87 ORF (without the termination codon) was ligated into the pBI221-GFP vector for the subsequent production of a green fluorescent protein (GFP)–tagged CmbHLH87 fusion protein. Polyethylene glycol was used during the transformation of Arabidopsis thaliana protoplasts with the recombinant plasmid (Lee et al., 2013 ). The subcellular localization of CmbHLH87 was determined based on the GFP signal, which was detected with the confocal fluorescence microscope (UltraVIEW VoX, Olympus, Japan) (Guo et al., 2019 (link)).

Freshly trypsinized HepG2 cells (1 × 10⁴−1 × 10⁵) were seeded in a 12-well plate and cultivated for 24 h with DMEM (Gibco, Thermo Fisher Scientific, Waltham, MA, USA). Cells were treated with EpoB and 1a at different concentrations (50 nM, 500 nM, and 5 μM) according to the IC₅₀ values for 24 h. The supernatants were removed, and the cells were washed with PBS buffer 2–3 times. The cells were further fixed with 4% paraformaldehyde for 1 h and then permeabilized with 0.2% Triton X-100 (diluted in PBS buffer) for 10 min. Next, the cells were blocked with bovine serum albumin (BSA) for 30 min and incubated with a primary antibody against β-tubulin (1:50, diluted in 2% BSA, β-tubulin Rabbit Monoclonal Antibody, Beyotime Biotechnology) for 1 h at room temperature. After that, the cells were treated with Alexa Fluor 488 goat anti-rabbit IgG (H + L) (secondary antibody, 1:500, diluted in 2% BSA, Beyotime Biotechnology) for another 1 h. The supernatants were removed, the cells were washed, and the nuclei were labeled with DAPI for 10 min. Images were collected using an Olympus BX81 fluorescence microscope system (Olympus, Tokyo, Japan) and a confocal microscope (UltraVIEW^®VoX).

Syncytial stage embryos were imaged on a Perkin Elmer ERS Spinning Disk confocal system (ERS software) mounted on a Zeiss Axiovert microscope, using a 63× 1.4 NA oil-immersion objective. Alternatively embryos were imaged on a Perkin Elmer Ultra-VIEW VoX (Volocity software) mounted on an IX81 microscope (Olympus), using a 60× 1.3 NA silicon immersion objective and an electron-multiplying charge-coupled device camera (ImagEM, Hamamatsu Photonics). All control/experiment pairs were analyzed on the same microscope system with identical illumination and acquisition settings.
For experiments to analyze the effects of PLP deletions, mRNAs encoding GFP-PLP deletions were injected into embryos and then imaged 60–120 min later.

Molm-13 and U937 cells were separately seeded into 6-well culture plates. After CKI treatment for 48 h, the cells were fixed in 4% paraformaldehyde (PFA) in 1 × PBS for 15 min at room temperature. The cells were first pre-treated and then blocked in 1 × PBS with 0.5% Triton X-100 and 5% BSA for 1 h. Next, the samples were incubated with anti-Prdx3 (1:100), Prdx2 (1:100), or Trx1 (1:100) antibodies overnight at 4 °C, followed by incubation with secondary antibodies conjugated to AF647 or CF568 (1:400 dilution). The samples were then analysed with a laser confocal microscope (OLYMPUS IX83, UltraVIEW VoX) with a 640 nm or 560 nm pulse. The images were collected with the Volivity software containing Acquisition, Quantitation, and Visualization modules.

Samples were imaged on a spinning disk confocal microscope (Perkin Elmer UltraView VoX) with a 60× 1.35 N.A. oil immersion UPlanSApo objective (Olympus) and a filter set to image fluorophores in DAPI, FITC, TRITC, and CY5 channels (center/bandwidth; excitation: 390/18, 488/24, 542/27, 632/22 nm; emission: 435/48, 594/45, 676/34 nm), the corresponding laser lines (488/4.26, 561/6.60, 640/3.2, 405/1.05, 440/2.5, 514/0.8 nm/mW), and an EMCCD camera (ImagEM, Hamamatsu Photonics). The camera pixel size is 8.34 µm, resulting in a pixel size in image space of approximately 139 nm. Optical sections were acquired with 200 nm spacing along the z-axis within a 512 × 512 pixel (71.2 × 71.2 µm) field of view.

The CmPR1 ORF was inserted into the pBI221-GFP vector to form a green fluorescent protein (GFP)-labeled CmPR1 fusion construst. Transient expression of the CmPR1-GFP fusion gene in Arabidopsis thaliana protoplasts was analyzed using polyethylene glycol method described by Lee et al. (2013) (link). GFP fluorescence was directly imaged using a confocal fluorescence microscope (UltraVIEWVoX, Olympus, Japan) under the excitation wavelength of 488 nm and the capture wavelength of 448–508 nm. Cell membrane and nucleus were detected by 1,10-dioctadecyl-3,3,30,30 -tetramethylindocarbocyanineperchlorate (Dil) and 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) staining, respectively.