Fv1000 confocal microscope

Cell growth was measured by both MTT assay and Trypan blue exclusion assay while the cell cycle, Annexin V-FITC based cell apoptosis, transwell-based cell migration, drug resistance assay, quantitative real-time PCR (qRT-PCR), and Western blot analysis was previously described [26 (link)]. Primers for qRT-PCR analysis (Table S3) and antibodies used for Western blot and Immunocytochemistry (ICC) analysis are listed (Table S4). For ICC analysis, cells were seeded on 20 × 20 mm cover slides and cultured in indicated medium. The cells cultured on cover slides were fixed with 4% paraformaldehyde (PFA) for 20–30 min and incubated with PBS in 4°C until use. The primary and secondary antibodies were sequentially applied onto the cells and DAPI was used as counter stain to define cell nuclei. Stainings were examined using Olympus FV1000 Confocal microscope or Zeiss LSM880 with AiryScan at Instrumentation Resource Center (IRC), NYCU. Final images were processed using Adobe Photoshop or PhotoImpact X3. Image J was used to quantify protein expression.

The yeast strains used for the FRAP experiment are diploids constructed by mating the haploid strains in BY4741 (MATa) and BY4742 (MATα) backgrounds. GFP tags were introduced into BY4741 and BY4742 by integrating the GFP–HIS3MX cassette into the C-terminus of the target genes in the native genome (Longtine et al., 1998 (link); Huh et al., 2003 (link)). See 'Key resources table' for strain information and Supplementary file 1 table S6 for the primer list. Yeast cells containing GFP-labeled factors were cultured to log phase in synthetic medium, and then transferred onto an agar pad and mounted by a coverslip. Cells were imaged using the 60X lens of a FV1000 confocal microscope (Zeiss) at room temperature. A 488 nm laser was used to excite and bleach green fluorescence. Depending on the GFP intensity, 3–10% laser power was used to take the images; 100% power was used to bleach the samples. The photobleaching time was set to 0.2–0.5 s, and the intervals between consecutive frames were 0.25–5 s. Two frames were acquired before photobleaching, followed by 28–48 frames afterwards. The bleached region covered ~10–25% of the nuclear region. Images were analyzed using Fiji-ImageJ. The average fluorescence intensities of the bleached and unbleached regions were recorded, and the ratio between them was used in the recovery curve.

Cells were fixed in 4% (wt/vol) paraformaldehyde for 15 min, washed with PBS and permeabilized with PBS containing 0.5% (vol/vol) Triton X-100 for 3 min. Cells were then washed with PBS, incubated with the indicated primary antibodies (anti-Flag antibody, #F7425; anti-SIRT1 antibody, #ab110304; anti-OGT antibody, #ab96718) overnight at 4 °C, washed with PBS and incubated for 45 min with the appropriate secondary antibodies (Alexa Fluor 488-conjugated goat anti-mouse IgG (H+L) secondary antibody, #A-11029; Alexa Fluor 594-conjugated goat anti-rabbit IgG (H+L) secondary antibody, #A-11012) at room temperature. After washing, coverslips were mounted using ProLong Gold mounting medium (Invitrogen). Images were acquired on either an Olympus FV1000 confocal microscope with a silicon immersion 60× objective or a Zeiss LSM 510 Meta with a silicon immersion 40× objective.

The cells grown on slides were fixed with 4% paraformaldehyde for 15 min and blocked with 1× phosphate buffer saline supplemented with 3 mg/mL bovine serum albumin, 100 mM glycine, and 0.25% Triton X-100 for 30 min. Subsequently, the slides were incubated with primary antibody rabbit anti-BDNF (1:500, ab108319), rabbit anti-light chain 3II (LC3II) (1:1000, ab48394), mouse anti-Tau (Tau-13, 1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and rabbit anti-BRUCE (5 µg/mL, ab19609) at 4 °C, followed by culture with the combination of the fluorophore and Alexa Fluor^® 647 secondary antibody (1:200, ab150075) at room temperature for 1 h. All the antibodies used above were provided by Abcam except Tau. Then, the cell slides were added with 4′ 6-diamidino-2-phenylindole for nucleus staining, then immersed in distilled water, dried and observed under a fluorescence microscope (Zeiss, Thornwood, NY) or FV-1000 confocal microscope.
For morphological analysis, the cells were fixed with 4% paraformaldehyde for 15 min and incubated with tau antibody (1:200, ab64193, Abcam) for 1 h, followed by another incubation with fluorophore combined with Alexa Fluor^® 647 secondary antibody (1:200, ab150075) for 1 h. The cells were then observed under the fluorescence microscope, and the length of the main axon in each cell was measured by five independent experiments.

Microscopic studies and photographs were carried out using Olympus FV1000 confocal microscope, ZEISS LSM 510 META confocal laser scanning microscope, or Nikon Eclipse 80i microscope (details were provided in related figure legends). DNA was stained by 4, 6-Diamidino-2-phenylindole (DAPI) (Sigma) at 1 mg/mL for 20 min before observation. Images were processed using Olympus Fluoview or Zeiss LSM Image Examiner. 3D analysis was accomplished in Olympus FV10-ASW Version 03.01.01.09. In time-lapse microscopy, filaments were cultured in a chamber slide filled with BG11 medium containing 0.5% agarose and imaged using Nikon Eclipse 80i microscope.