Tcs sp5 2 aobs confocal microscope

Oligonucleotide primers (Forward: 5’-CGAAGATCTCTAATACGACTCACTATAGGGC-3’, Reverse: 5’-AGCAAGTTAAATAAGCTGGTGTCAAAAATAGAC-3’) were synthesized and used to amplify BRLF1 gene by RT-PCR. The BRLF1 cDNAs were purified using a QIAquick PCR Purification Kit (Qiagen), cleaved with the restriction endonucleases (BglII and HindIII), and cloned into pmCherry-N1 Vector (Clontech). The capped sense BRLF1-mCherry and CaaX-EGFP mRNA (to label membrane with EGFP) were respectively transcribed by using the mMessage mMachine T7 kit and SP6 kit (Life Technologies). The synthesized mRNAs with 2.3 nl of 200 ng/μl BRLF1-mcherry and 120 ng/μl Caax-EGFP were dissolved in 0.2% phenol red and then microinjected into Tg (h2afva:h2afva-GFP) embryos (From Taiwan Zebrafish Core Facility at National Health Research Institutes, Zhunan, Taiwan) at one cell stages using an IM 300 Microinjector (Narishige). After 24 h, embryos were anesthetized using 0.4% tricaine and embedded in a 1% low-melt agarose. The live images of cell mitosis in eyes of embryo were visualized by the Leica TCS SP5II AOBS Confocal Microscope and recorded using the digital camera.

HEK-293 T cells were transfected with vector plasmids or Flag-nsp14 expression plasmids. At 24 hpt, cells were stimulated with TNF-α for 30 min and fixed with 4% paraformaldehyde. Cells were then permeabilized and incubated with appropriate antibodies as previously described (Nagendraprabhu et al. 2017 (link)). The nuclei were stained with DAPI (4′6´-diamidino-2-phenylindole) (Sigma) for 10 min. The stained cells were analyzed using a Leica TCS SP5 II AOBS confocal microscope at the appropriate settings.

DNA fragmentation was detected in situ on cells seeded on poly-L-lysine-coated glass slides (12 mm diameter) with the Click-It^® Plus Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) Assay (Molecular Probes Life Technologies) following the manufacturer’s instructions. Nuclei were stained with 0.1 μg/ml 4',6-diamidino-2-phenylindole (DAPI) in PBS for 10 minutes at room temperature. Then, slides were mounted in 90% glycerol/10% PBS, sealed and imaged with a Leica TCS SP5II AOBS confocal microscope equipped with a HCS PL APO CS 63x/1.4 oil immersion objective. DAPI was excited at 405 nm (diode laser) and emission was detected in the 445–490 nm range; Alexa Fluor^® 647 picolyl azide (TUNEL signal) was excited at 633 nm (HeNe laser) and emission was detected in the 645–750 nm range. To obtain the TUNEL signal normalized for the cell density, fluorescence intensity in the emission window of TUNEL was expressed as levels of gray, subtracted for the background fluorescence and normalized for the background-subtracted fluorescence intensity in the emission window of DAPI. Cells left untreated or treated overnight with 2 μM staurosporine were considered as the negative and the positive control, respectively. In the positive control, a TUNEL signal was detected in 100% of nuclei.