Sp5 laser confocal microscope

Cells fixed in methanol at −20°C were washed with phosphate-buffered saline (PBS), mixed with 2% bovine serum albumin and 0.5% Triton X-100 for blocking, and subsequently incubated with primary antibodies for 2 h. After washing, the cells were incubated with secondary antibodies and DAPI for 45 and 1 min, respectively. Coverslips were mounted using ProLong Gold anti-fade (Invitrogen), and images (63×) were captured using a Leica SP5 laser confocal microscope. Alternatively, cells fixed in 4% paraformaldehyde were washed with PBS, and primary antibodies were added and incubated overnight. After washing, the cells were incubated with secondary antibodies and DAPI for 1 h (1% BSA and 0.3% Triton X-100) and 1 min, respectively. The rest of the steps are identical, as mentioned above.

Polarized epithelial monolayers were fixed with 4% paraformaldehyde for 20 min, permeabilized with 0.05% Triton X-100, and washed three times with PBS. To determine disruption of TJs, we immunostained cells with mouse monoclonal antibodies against ZO-1 (Invitrogen/Life Technologies, Austin, TX) for 1 h at room temperature. For detection of AJs, polarized cells were stained with rabbit antibodies to E-cadherin and mouse monoclonal antibodies to nectin-1 (both from Invitrogen/Life Technologies). To determine HSV-1 infection, we immunostained infected cells with mouse antibodies against HSV-1 ICP4 and gB (both from Santa Cruz Biotechnology, Inc., Dallas, TX), rabbit anti-gD (HSV-1) (MyBioSource, San Diego, CA), and goat anti-HSV-1/2 antiserum (AbD Serotec, Raleigh, NC). All antibodies were diluted 1∶50 in blocking solution (3% BSA and 0.03% saponin in PBS). Monolayers were washed and then incubated for 25 min with FITC- or TRITC-labeled secondary antibodies (Vector Laboratories, Inc., Burlingame, CA). Cell nuclei were counterstained with TO-PRO-3 (blue) or propidium iodide (red) (Molecular Probes/Life Technologies). Cells were analyzed by using a Leica SP5 laser confocal microscope.

Cells were fixed with 4% ice-cold paraformaldehyde (PFA) (Sigma) for 15 min and permeabilized with 0.1% Triton X-100 (Sigma) in PBS (Gibco) for 10 min, followed by 30 min of blocking in 1% BSA (Roche). The cells were then stained overnight at 4 °C with the following primary antibodies: sarcomeric α-actinin (1:400; Sigma), LRP6 (1:100; Abcam), LRP5 (1:100; Abcam), Cx43 (1:200; Sigma), PDI (1:100; Novus), GM130 (1:100; CST), α-tubulin (1:400; Sigma), Cx45 (1:200; Abcam), Cx40 (1:200; Abcam) and β-catenin (1:100; Sigma). Next, the cells were washed with PBS and incubated for 1 h with the respective secondary antibodies conjugated to Alexa Fluor 488/555/633 (1:300; Invitrogen), after which the cells were stained with ToTo-3 (Invitrogen) for an additional 20 min if needed.
Sections of mouse and rat hearts were deparaffinized and rehydrated, and then boiled in sodium citrate solution for 20 min for antigen retrieval. Slides were processed for immunofluorescence visualization according to the procedure used for cultured cardiomyocytes. Images were captured using a Leica SP5 laser confocal microscope with a × 63 oil lens. Co-localization was analysed using Image-Pro Plus software (MediaCybernetics).

Tubulin (15 μM) was polymerized in BRB80 buffer (80 mM Pipes, 1 mM EGTA, 1 mM Mgcl2 pH 6.9) in the presence of 10% DMSO, 15 μM Taxol, and 1 mM GTP at 35 °C for 15 min. Aliquots of polymerized MTs were incubated with EB1 (1 μM) for 5 min followed by incubation with WT Ska1 or Ska1 ΔSHLP (0.5 μM) for another 15 min at room temperature. The MT-protein mixtures were then fixed with 1% glutaraldehyde in BRB80 for 5 min at room temperature followed by diluting 50 times in BRB80 buffer prior to layering on a 15% glycerol cushion and sedimentation onto 0.1% poly-L-lysine–coated coverslips. Coverslips were blocked with 1% BSA-BRB80 for 30 min and incubated with mouse monoclonal α-tubulin (Sigma), rat monoclonal EB1 (Abcam), and rabbit polyclonal Ska1 antibodies (Novus) for 45 min followed by incubation with secondary antibodies, anti-mouse Alexa 555, anti-rat Alexa 488, and anti-rabbit Alexa 647. Images were captured using Leica SP5 laser confocal microscope. The fluorescence intensities of the proteins associated with MTs were analyzed using Leica LAS AF lite software. The per-pixel intensity of Ska1 bound to MTs was quantified by drawing line region of interest of 2 μm length on the MTs (∼120 in number in three experiments) after background subtraction.

Adult flight muscles were dissected in DSM. Hemithoraxes were incubated in staining solution (5 μM MitoSox™ Red [M36008, Thermo Fisher Scientific] and 100 nM MitoTracker Green [M7514, Thermo Fisher Scientific]) for 12 min at room temperature. The hemithoraxes were washed twice with DSM for 30 s at room temperature. Samples were mounted in DSM solution. Slides were then imaged on the Leica SP5 laser confocal microscope using identical settings for each condition. MitoSox intensity was quantified using Image J.

Adult flight muscles were dissected in Schneider's medium (DSM) and then incubated in 2 µM JC‐1 solution (diluted in DSM) for 45 min at room temperature. The muscles were then immediately mounted in DSM and imaged using the Leica SP5 laser confocal microscope. The intensity of fluorescent aggregates was quantified using ImageJ.

Cells fixed in methanol at − 20 °C were washed with phosphate-buffered saline (PBS), mixed with 1% bovine serum albumin and 0.1% Triton X-100, and subsequently incubated with primary antibodies for 2 h. After washing, the cells were then incubated with secondary antibodies and DAPI for 45 and 1 min, respectively. Coverslips were mounted using ProLong Gold anti-fade (Invitrogen), and images (63X) were captured using a Leica SP5 laser confocal microscope.