Lsm 780 fluorescent microscope

For imaging of lysosome trafficking mediated by extracellular pH, Hela, and MiaPaCacells stably expressing mCherry-GFP-LC3were transfected with scrambled siRNA(CTL) or REP1 siRNA following the reverse transfection protocol, and incubated at 37 °C for 24 h in an 8-well Lab-Tek chamber. Subsequently, cells were changed with pH7.4 and pH6.8 DMEM media for 16 h. Then, nuclei were stained using DAPI. For lysotracker staining, LysoTracker Red was treated on the cells 30 min prior to imaging. Images were captured using an LSM780 fluorescent microscope (Zeiss, Oberkochen, Germany) and quantified using the software ZEN (Zeiss, Oberkochen, Germany). Auto lysosome signals (Red) from mCherry-GFP-LC3 were captured in at least five distinct fields from different regions for an individual experimental set. The total puncta area per cell was normalized by the area of DAPI-stained nucleus of that cell.

TZM-bl and HEK293T cells were seeded on poly-L-lysine coated glass-bottom µ-Dishes (Ibidi GmbH, Martinsreid, Germany). 24 h later the cells were washed with PBS and fixed with 2.5% paraformaldehyde for 10 min. The cells were washed twice with PBS, permeabilized for 10 min with 0.5% Triton X-100, washed three times with PBS, and blocked for 1 h with blocking buffer (1× PBS, 1% glycine, 0.1% Triton X-100) at room temperature. Then the cells were incubated for 1 h with 2F5 (10 µg/mL) diluted in blocking buffer, then rinsed three times in PBS with 0.1% Triton X-100 and stained with anti-human Alexa Fluor 488 conjugates (Invitrogen) diluted 1:200 in blocking buffer. Cells were left for 1 h at room temperature in the dark, rinsed three times with blocking buffer, and stained with DAPI as proposed by the manufacturer (NucBlue, Molecular Probes, Eugene, OR, USA). Images were captured using an LSM 780 fluorescent microscope (Carl Zeiss, Oberkochen, Germany) with an ×40 oil emersion lens. The LSM 5 Image Examiner software was used.

The PLA was performed with a DuoLink® PLA kit (number DUO92101, Sigma-Aldrich), and following the manufacturers' protocol. In brief, after blocking and incubating with mouse anti-DYRK1A and rabbit anti–IRS-1 primary antibodies, the slips were washed and then incubated with PLA probes. The probes were then ligated by the ligation reagent. Next, the amplification reagent containing polymerase and oligonucleotides labeled with detectable fluorophores was applied. After wash, cells were incubated with Alexa Fluor® 488 conjugate α-tubulin (number 5063, Cell Signaling). Finally, the slips were washed and mounted with Duolink® in situ mounting medium containing DAPI. For negative controls, IRS-1 antibody was substituted with anti-rabbit IgG antibody, or DYRK1A antibody substituted with anti-mouse IgG antibody. Z-stack images were captured using LSM 780 fluorescent microscope (Carl Zeiss, Jena, Germany) and the maximum intensity projections were obtained by ZEN software.

After incubation in the dark at 23°C for 12–24 h, the fluorescence of GFP or GFP-protein fusions was viewed under an LSM710 confocal laser scanning microscope (Carl Zeiss, Inc.). Transformation efficiency of the protoplasts was determined based on the GFP-reporter expression using the transient expression vector pAN580-GFP. GFP fluorescence was observed and 3–5 images were taken randomly under an LSM780 fluorescent microscope (Carl Zeiss, Inc.) or LSM710 confocal laser scanning microscope. The transformation efficiency was measured as a bright green fluorescent protoplast number in view/total protoplast number in view (%). At least three photographs were taken for each sample, and these experiments were independently conducted at least three times. For subcellular localization analysis, plant organelle markers pGreenII62-SK-AtWAK2-GFP and pGreenII62-SK-AtPIP2A-GFP, and the control vector pGreenII62-SK-GFP were transfected into orchid leaf base protoplasts. Red chlorophyll fluorescence was used to indicate the intercellular location of chloroplasts.