Alexa 546

Larval brains were dissected in PBS and fixed for 20 min at room temperature in 4% formaldehyde and fixation buffer (PBS, 5 mM MgCl2, 0.5 mM EGTA). After fixation, brains were rinsed and washed in 0.3% PBS Triton X100 (PBT). Samples were blocked with 10% normal goat serum (NGS) in 0.3% PBT at room temperature and incubated with the primary antibodies overnight at 4°C. Brains were then washed in 0.3% PBT and incubated with the secondary antibodies overnight at 4°C. Brains were washed in 0.3% PBT and mounted in Vectashield (Vector Laboratories, Burlingame, CA, USA). The following primary antibodies and dilutions were used: guinea pig anti-Dpn (1:10,000) and rat anti-L’sc (1:5,000) (Caygill and Brand, 2017 (link)), chicken anti-GFP (1:2,000) from Abcam, mouse anti-Delta (1:50, C594.9B) from DSHB and mouse anti-Notch (intracellular domain, ICD) (1:50, C17.9C6) from DSHB. Fluorescently conjugated secondary antibodies Alexa405, Alexa488, Alexa546 and Alexa633 (all 1:200) from Life Technologies.
Images were acquired with a Leica TCS SP8 confocal microscope (Leica Microsystems, Wetzlar, Germany) and analysed with Fiji (Schindelin et al., 2012 (link)). Figures and illustrations were assembled using Adobe Photoshop CS3 and Adobe Illustrator CS3 (Adobe Systems, San Jose, CA, USA).

The tissue sections were prepared as earlier described. The tissue was encircled with a PAP pen and blocked for 1 h with Ultra V Block (TA-060-UB, Thermo scientific). Approximately 25 µg antibody fragments were dissolved in Ultra V Block, 10 % goat serum and 1:100 anti-CK19 to a total volume of 100 µL and added to the encircled area. Incubation was performed for 3 h in humid chambers. The liquid was removed by aspiration, and the slide was washed four times 1 min in PBS. The slide was incubated for 30 min in the dark with mouse Cy3-conjugated anti-c-Myc antibody [9E10] 1:250 (Sigma-Aldrich), Alexa Fluor 488-conjugated goat anti-mouse IgG2a 1:500 (Invitrogen), DAPI 1:1000 (Invitrogen) and 10 % goat serum dissolved in Ultra V Block to 100 µL. Alternatively, the antibody fragments above were replaced with an anti-Ki67 antibody (Abcam) in a dilution of 1:100, and as secondary antibody, a goat anti-rabbit antibody coupled to Alexa 546 (Life technologies) was added in dilution of 1:500. The slide was washed three times 1 min in PBS and mounted with Fluoromount mounting media (Sigma-Aldrich) and cover glass.

To analyze functional coupling between transduced SkM, single-cell-electroporation was used. Sharp electrodes, filled with 40 mM KCl and two different dyes (Alexa350 and Alexa546-Dextran) were attached to the cell membrane of cultured (10–20 days) myoblasts resulting in a resistance of 140–190 MΩ. Alexa350 (349 Da; 1 ng/nl, Life Technologies, Darmstadt, Germany) emits blue fluorescence and is a small molecule, which is able to pass through Cx43 gap-junctions between adjacent cells. Dextran coupled Alexa546 (10 kDa; 10 ng/nl, Life Technologies, Darmstadt, Germany) emits in the red region of the spectrum and diffuses only to neighbouring cells through cytoplasmic bridges because of its high molecular weight^{49 (link)}. Next, the cell membrane was perforated by applying an alternating current of 400 Hz and 10–50 nA in square-pulses and the electroporated myotubes were allowed to load via iontophoresis with the two different dyes. Dye transfer to adjacent EGFP⁺ cells was observed using fluorescence microscopy (Axiovert 200, Carl Zeiss, Jena, Germany) in 28 EGFP⁺ cells, dye transfer into an EGFP^-SkM was never observed.

Rabbit polyclonal antibodies (pAbs) for ERC1, FLAG, FN, LL5β (recognizing human and monkey LL5β, but not mouse LL5β), and mouse monoclonal antibodies (mAbs) for ERC1a (clone ELKS-30), tubulin, FLAG clone M2 (Sigma-Aldrich); rabbit pAb for GFP (Life Technologies); chicken pAb for GFP and rabbit pAb (Abcam); rabbit pAb anti DS-RED (Clontech); rabbit pAb for Liprin-α1 (Protein Tech); mouse mAb for filamin A (Millipore), paxillin (clone 349, BD Biosciences), GST (Amersham Biosciences), His-tag (Qiagen), LL5α/β (clone 1H12) [19 (link)]; hamster mAb for LL5α [5 (link)] was kindly provided by dr. Yuko Mimori-Kiyosue (RIKEN Center for Biosystems Dynamics Research, Kobe, Japan). Secondary Abs Alexa-488, Alexa-568, Alexa-546 and Alexa-647, phalloidin Alexa-568 and Alexa-647, and Oregon green 488-gelatin (Life Technologies); HRP-conjugated anti-rabbit and anti-mouse secondary Abs (Jackson and Amersham Biosciences). Other reagents included: FN (Corning); poly-L-lysine hydrobromide.

Mice were perfused with Ca²⁺/Mg²⁺ free Tyrode’s solution followed by 4% paraformaldehyde with 0.4% picric acid in 0.16 M phosphate buffer. The brains were dissected, postfixed for 2 hours, and equilibrated with 10% sucrose. Brains were frozen and cryo-sectioned to obtain 14 (for the striatum) or 20 (for the midbrain) μm thick sections. After 1 hour in blocking solution (PBS+ 0.3% TrItox X-100+ 1% BSA), the tissue sections were immunolabelled overnight with primary antibodies against TH (1:500, Pel-Freez; 1:1000, Chemicon), IBA1 (1:1000, Wako), GFAP (1:1000, Abcam) and CD45 (1:100, Serotec). For fluorescent staining, Cy3- (1:400, Jackson Biolabs), Alexa 546- (1:400, Life Technologies) and Alexa 633- conjugated secondary antibodies (1:400, Life Technologies) were used. Confocal images were acquired by sequential scanning using a LSM800 or LSM880 microscope (Zeiss). Relative intensity of mitoYFP-labelled mitochondria in the striatum and IBA1, CD45 and GFAP immunoreactivities in the midbrain were quantified using Fiji software. Confocal images were thresholded and total intensity was measured using automatic particle counting.
Live microscopy on sorted cells was performed as previously described [43 (link)].

The following primary antibodies were used for IHC or western blot analyses: mouse anti-GPR56 (H11) (1:200)40 (link) and rabbit anti-GPR56 (199) (1:200)19 (link), rabbit anti-MBP (Millipore; Cat #AB980, 1:200), rat anti-MBP (Abcam, Cat# ab7349), mouse anti-O4 (Millipore; Cat #MAB345, 1:400), rabbit anti-NG2 (Millipore; Cat #AB5320, 1:200), goat anti-Sox2 (Santa Cruz; Cat #sc-17320, 1:400), rabbit anti-Olig2 (kind gift from Charles Stiles, 1:10,000), rat anti-PDGFRα (BD Bioscience; Cat #558774, 1:500), rabbit anti-PDGFRα (Cell Signaling Technologies; Cat #3164S, 1:500) and rat anti-Ki67 (Affymetrix eBioscience; Cat #14-5698-80, 1:100), rat anti-BrdU (Accurate Chemical and Scientific Corporation; Cat #OBT0030S, 1:100), rabbit anit-PLP (Abcam, Cat #ab28486, 1:1,000), mouse anti-RhoA (Cytoskeleton, Cat# ARH03-A, 1:500), mouse anti-CDK2 (Santa Cruz; Cat #sc-6248, 1:1,000), mouse anti-β-actin (Sigma, Cat #A5044, 1:5,000) and mouse anti-Ki67 (BD Bioscience; Cat #550609, 1:100). Secondary antibodies were goat anti-mouse or anti-rat conjugated with either Alexa 488 (Life Technologies, 1:1,000) or Alexa 546 (Life Technologies, 1:1,000) and goat anti-rabbit conjugated with Alexa 546 or 555 (Life Technologies, 1:1,000), goat anti mouse or rabbit IgG-HRP (Sigma, Cat# A4416 or A6154, 1:3,000).

Monocytes, plated on 24 mm glass coverslips (Marienfeld, Germany), were incubated at 37 °C in the presence of LPS or LRZ for 6 h (N = 6). Samples were then washed with PBS, fixed and permeabilized with 4% paraformaldehyde and 0.5% TritonX-100 in PBS. After washing and saturation with PBS supplemented with 2% (w/v) BSA (Sigma), cells were labeled with primary antibodies against IL-1β (mAb 3ZD) and LAMP2A (rabbit, Abcam, UK) in PBS/BSA, followed by incubation with specific secondary antibodies labeled with Alexa-546 (Life Technologies, USA) or Alexa-647 (Invitrogen, USA). Images were obtained using a Leica TCS SP8 scanning confocal microscope equipped with a 63 × /1.4 oil objective and LAS AF software with a Z-stack of 170 nm. Reconstructed 3D volume was obtained using ImageJ. For TIRF analyses, cells were examined under a Leica SR Ground State Depletion (GSD), microscope, with a 160 × /1.45 objective and an sCMOS camera (Hamamatsu Flash ORCA 4.0). In order to enhance the signal vs. background and identify/quantify the vesicles, images were filtered with a Laplacian of Gaussian filter (using the FeatureJ-Laplacian plugin of ImageJ with a smoothing scale parameter of 1), before applying a histogram based threshold.