Lsm 710 microscope

Yeast cells were cultured in YPD or selective medium until OD₆₀₀ of 1.0. After washing with cold phosphate buffered saline (PBS), cells were fixed with 4% formaldehyde in PBS for 30 min and treated with DAPI (Solarbio, c0065) for 15 min at room temperature. Cells were then washed with cold PBS. The cell morphology was visualized using a ZEISS LSM710 microscope (Germany) with a 100× oil immersion objective by fluorescent microscopy. Images were acquired using ZEN Imaging Software ZEN 2.1 (ZEISS). DAPI was used to indicate the nucleus.
For immunofluorescence, cells were grown to OD₆₀₀ of 1.0 and fixed with formaldehyde for 30 min. After fixation, cells were resuspended in 0.5 ml of 1.2 M sorbitol phosphate citrate buffer (SPC) containing 0.01 % zymolyase 20 T for 1.5 hr at 30 °C. The spheroplasts were added on the poly-L-lysine-coated slides, blocked by 3% bovine serum albumin (BSA) in phosphate buffered saline (PBS) at 30 °C for 1 hr and then incubated with primary antibodies at 4 °C overnight. After washing with PBS for three times, cells were incubated with Alexa Fluor 594 goat anti-rabbit IgG (Invitrogen) at 30 °C for 1 hr. Cells were then washed with cold PBS and visualized by ZEISS LSM710 microscope using ZEN Imaging Software (ZEISS). The merged color images were generated by Fiji software.

The phase separation assays were performed as previously described^{54 (link)}. The proteins and DNA used for droplet formation were adjusted to the desired concentrations in the phase separation buffer composed of 25 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 10% PEG-8000. Then 3–6 μl of the mixed sample was deposited onto a microscope slide. Imaging of the droplets was captured by a Zeiss LSM 710 microscope (Zeiss, Germany).
The fluorescence recovery after photobleaching (FRAP) experiments was performed on a Zeiss LSM 710 microscope with a 63x oil objective, following the procedure previously described^{55 (link)}. In our in vitro FRAP experiment, the region of interest (ROI) (diameters of 4 to 8 μm) was bleached for 300 iterations using a laser with 488 nm wavelength at normal 100% laser transmission. Then the post-bleach time-lapse images were collected every 10 s for 5 min. The mean fluorescence intensities from the photobleached region of every time-lapse image were measured and recorded. The raw data were processed and plotted by GraphPad Prism.

The number of axonal terminals perforating PNNs was quantified in 33 × 33 × 5 µm z-stacks obtained with high-resolution confocal microscopy using the LSM 710 microscope (Zeiss, 100 × alpha Plan-Apochromat objective, NA 1.46, voxel size 60 × 60 × 500 nm). ROIs were positioned in the motor cortex L5 (see Additional file 4: Fig. S1) containing a single PNN-coated neuron. Synaptic terminals expressing VGAT or VGLUT1 that associated with WFA labeling were counted using an automated ImageJ routine (see Additional file 1). In each animal, four ROIs obtained from two adjacent brain sections were analyzed.
The surface of microglia/macrophage-neuron contacts was quantified in 75 × 75 × 10 µm z-stacks obtained with high-resolution confocal microscopy using the LSM 710 microscope (Zeiss, 63 × alpha Plan-Apochromat objective, NA 1.4, voxel size 70 × 70 × 450 nm). ROIs were positioned in the motor cortex L5 containing a single PNN-coated neuron. Surfaces representing IBA1 (microglia/macrophages) and Kv3.1 (fast-spiking interneurons) labeled cells were generated by automated thresholding with IMARIS 9.9 software (Oxford Instruments, Stockholm, Sweden) using the standard surfaces function. The area of contact between cells was quantified as the intersection between IBA1 and Kv3.1 surfaces.