Lsm880 airyscan confocal laser scanning microscope

Cells (2 × 10⁴) were seeded in 35 mm glass bottom culture dishes (Thermo Fisher Scientific) and incubated overnight. After fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and blocking with 3% bovine serum albumin, the cells were stained with primary antibodies overnight at 4 °C, followed by incubation with Alexa Fluor 647-conjugated or CY3-conjugated secondary antibodies (Beyotime) for 1 h. The dishes were counterstained for cell nuclei with DAPI (Beyotime). Fluorescent images were acquired using an LSM880 + Airyscan confocal laser scanning microscope (Carl Zeiss, Oberkochen, Germany).

Cultured H2 bacterial cells were thoroughly washed and subjected to click labeling with the biotin tag. Streptavidin Alexa 647 was then used to stain the cells. Fluorescently labeled bacterial cells were spotted on a glass slip, airdried for 15 min, and covered with a cover slip with ~ 1 mm thickness. Confocal microscopy was performed on a Zeiss LSM 880 Airyscan confocal laser scanning microscope. Imaging analyses were processed with FlowJo_v10.

Protocol adapted from Tampere and Mortusewicz, 2016 (link). Briefly, U2OS cells were plated in a 35 mm μ-Grid dish (Ibidi) a day before microirradiation. Cells were sensitized by 1 μg/ml Hoechst 33342 (Invitrogen) for 10 min and transferred to the stage incubator of LSM880 Airyscan confocal laser scanning microscope (Zeiss). Using the bleaching mode in ZEN software, DNA damage was induced by irradiation of a 5-pixel wide region with a 405 nm diode laser set to 15% power, 1 iteration, zoom 1, averaging 1, pixel dwell time 0.27 μs (speed 7). Cells were fixed 1 hour after the irradiation and stained with anti-phospho-Histone H2A.X (Ser139) and anti-D tag antibodies following the immunofluorescence protocol.

Fluorescence recovery after photobleaching (FRAP) experiments were performed using a Zeiss LSM 880 Airyscan confocal laser scanning microscope (Zeiss, Oberkochen, Baden-Württemberg, Germany) with a C Plan-Apochromat 63×/1.4 Oil DIC M27 objective, 488 nm Argon Laser, and QUASAR detector at 495–571 nm.
Samples were incubated for at least 1 h on a Chamber Coverglass System with a BSA coating (Cellvis, C8-1.5H-N T) in 25 mM HEPES buffer pH 7.5, 75 mM NaCl, and 5% PEG-4000. A circular region of interest (ROI) with a one-third diameter relation of droplets settled at the bottom of the cover glass was bleached using 90% laser power. Droplets of an approximate diameter of 3 μm were selected for homotypic and heterotypic condensates, respectively (n = 7). A z-stack of three images was taken in two blocks of 90 cycles of 300 ms and 5 s, respectively, recording fluorescence intensity for three different ROIs (bleached droplet, reference droplet, and background). Acquired images were 196 × 196 pixels, with 0.09 μm × 0.09 μm pixel size, 16-bit, 3 slides with 0.8 μm of section, and 1.35 μsec pixel dwell. Fluorescence intensities from bleached ROI were corrected for photofading and normalized to the bleaching depth as described in [36 ]. Fluorescence recovery data were evaluated using the Fiji software (2.0.0-RC-68/1.52G).

1 × 10⁴ N2a^eGFP and N2a^GPR37-eGFP cells were seeded in an 8-well chamber with a cover slide bottom (Nunc, ThermoFisher, Waltham, MA, USA). Cells were washed in ice-cold HBSS, and CtxB-Alexa 647 in 0.1% BSA was added for 5 min in line with published protocol for labeling the plasma membrane fraction of GM1 [35 (link)]. Cells were washed twice in PBS and fixed in 4% PFA for 10 min. Cells were imaged in a Z-stack using Zeiss LSM 880 Airyscan confocal laser scanning microscope using a Plan-Apochromat 20x/0,8 objective. ImageJ was used to merge Z-stack, and the fluorescence intensity at the plasma membrane was quantified in single eGFP-positive cells in order to correctly quantify the entire plasma membrane. In total, 53 cells from three separate experiments were quantified for each condition and normalized to fluorescent intensity of N2a^eGFP.

Treated cells were harvested using trypsin and seeded on slides the day before immunofluorescence assays. The next day, cells were fixed with 4% formaldehyde, followed by the treatment with 1% Triton X-100 (Sigma, St. Louis, MO, USA) for permeabilization and then blocked with 2% bovine serum albumin for 40 min at room temperature. To visualize p-CREB, coverslips were incubated with an anti-p-CREB antibody overnight and then incubated with a FITC-conjugated secondary antibody (Cell Signaling Technology) for 1 h. DAPI (Sigma) was included during staining to visualize the nuclei of cells. Images were captured using a LSM880 + Airyscan confocal laser scanning microscope (Carl Zeiss, Oberkochen, Germany) equipped with a 20X objective (NA 0.8). The fluorescence intensity was analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA) based on previously described procedures^{21 (link)}.

Images were taken with a ZEISS LSM 880 Airyscan confocal laser scanning microscope equipped with ZEN2.1 software, using a Plan-Apochromat 63×/1.4 Oil DIC M27 63× oil objective. Preparation of images and quantification of the immunofluorescence (IF) was done using Image J (National Institutes of Health, NIH, http://rsb.info.nih.gov/ij/) and OpenView software [57 (link)]. Firstly, the background was subtracted from all channels using Image J, and the synaptic IF intensities were assessed in a region of interest (along 20 µm of the proximal dendrite) which was set by the mask in the channel for synaptophysin. This mask was created semi-automatically by using OpenView. The final adjustment of images was made by ImageJ and Photoshop (Adobe Systems, San Jose, CA, USA).