Lsm780

Paraffin-embedded brain sections were dewaxed and antigen retrieval was performed as described previously (56 (link)). The following primary antibodies were used for immunolabeling: a chicken polyclonal anti-MAP2 antibody (ab5392; Abcam), a rabbit monoclonal anti-Na,K-ATPase α subunit antibody (ab76020, Abcam), and a mouse monoclonal anti-Na,K-ATPase α1 subunit antibody (a6F; DSHB). Confocal microscopy was performed with Zeiss LSM 780 and Leica TCS SP8 microscopes. For further details, see Supporting information (Text S3).

A 25–50 µg of TX-100 soluble material was separated by 3–15% gradient or 12% SDS–PAGE prior immunoblotting and chemiluminescence detection (Pierce). Antibodies used in this study were anti-RUY4 raised in rabbit against peptides 375–391 and 454–470 of mouse RUFY4. Mouse Anti-Myc (9B11, Cell Signaling), mouse Anti-Flag (M2, Sigma), rat anti-LAMP1 (134B, Biolegend), mouse anti-AIF (E-1, Santa Cruz), mouse anti-SDHA (2E3GC12, Abcam), mouse anti-LC3 (2G6, NanoTools), mouse anti-ß-actin (AC-15, Sigma). Secondary antibodies were from Jackson Immunoresearch, Molecular Probes (USA) and from Cell Signaling Technology. For immunofluorescence, cells on coverslips were fixed with 3.5% paraformaldehyde and permeabilized with 0.1% Triton X-100. Images were taken by a Zeiss LSM780 or Leica SP5 confocal microscope using 63× or 40× objective. Processing and quantification was performed using Fiji software [37 (link)]. Co-localization was quantified using JACoP plugin [38 (link)]. Statistical analysis was performed using Graphpad Prism. For two sets of values, we used t-tests, for multiple sets of values one-way ANOVA. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. MitoTracker DeepRed and MitoTracker Green staining was performed according to the manufacturer's instructions (Thermofhisher) and detected by flow cytometry.

Confocal laser scanning microscopy was performed using Carl Zeiss(Germany) LSM710, Carl Zeiss LSM780 and Leica (Germany) TCS SP8 confocal microscope. Images were processed using Imaris (Bitplane), ImageJ, and Adobe Photoshop software.

Cells for imaging were grown in 24-well plates on coverslips. Samples were fixed with 4% (vol/vol) electron microscopy-grade formaldehyde (Polysciences) for 10 min and then permeabilized with 0.1% Triton for 5 min. Cells were then incubated with blocking buffer (2% [vol/vol] FBS in PBS), with the addition of 100 μg/ml human IgG when rabbit primary antibodies were used, for 30 to 60 min and then with primary antibodies. Secondary antibodies used were anti-rat, anti-rabbit, and anti-mouse isotype-specific AlexaFluor 488, 568, or 647. Coverslips were mounted on microscope slides by using ProLong Gold antifade reagent with 4′,6-diamidino-2-phenylindole (DAPI; Thermo Fisher). All images were acquired with a confocal microscope (Zeiss LSM780 or Leica SP5).
For cell morphology assays, HFF-Tert cells grown in 6-well dishes were infected at 5 PFU/cell and incubated for 16 h. Cells were fixed with 4% (vol/vol) formaldehyde for 20 min, washed with PBS, and then incubated with anti-gD (LP2). Samples were then washed in PBS, incubated with AlexaFluor 488 secondary antibody, incubated with PBS containing DAPI, washed with PBS, and then stored in PBS. Images were acquired using the 10× lens of an Olympus IX81 wide-field fluorescence microscope and the Image-Pro Plus software (Media Cybernetics).

Zeiss laser scanning confocal microscope LSM710, LSM780 and Leica laser scanning microscope SP8 containing laser lines at 488, 561 and 633 nm were used to image immunostained fixed or live embryos. The 40X objective having NA 1.4 of these microscopes was used for imaging. The laser power, scan speed and gain were adjusted with the range indicator mode such that 8-bit image acquisition was in 0–255 range. For both fixed or live imaging, an averaging of 2 was used during image acquisition. Optical sectioning of 1.08 μm and 0.68 μm was used to acquire images at Zeiss and Leica confocal microscopes, respectively.

Images of cell nuclei were registered using a Zeiss LSM 780 (and Leica SP5) confocal microscope with either 40× or 63× water immersion Plan Apo objective lens (NA = 1.2). The Zeiss system was equipped with two primary long-pass dichroic mirrors (405 and 488 nm) and a 32-channel GAsP PMT working in integration mode. The Leica system was equipped with an acousto-optical beam splitter (AOBS) and multialkali single-channel PMTs. Fluorescence of H1-eGFP was excited with 488 nm light (40 mW Ar ion laser at 1 % power, unless stated otherwise) and detected in the 490–560-nm range, whereas the fluorescence of DAPI was excited with 405 nm (20-mW diode laser) and detected in the 410–480-nm range. Unless otherwise indicated, the images (optical sections) were registered at 16-bit precision with a pixel size of 0.063 nm (256 × 256 pixels), pixel dwell time of 5.09 µs and the confocal pinhole set to 1 airy unit (at 530 nm). The imaging of live cells was performed at 37 °C in DMEM, with 5 % CO₂. Fixed cells were imaged at room temperature.