Goat anti mouse igg2b alexa fluor 647

NRVMs were washed with phosphate buffered saline (PBS) and fixed with 4% paraformaldehyde at room temperature for 10 min. Samples were further washed three times with a PBS solution and permeabilized with a PBS solution containing 0.3% Triton X-100 for 30 min. Then, samples were blocked for 30 min using a PBS solution containing 2% fetal bovine serum (FBS, Sigma Aldrich, #F2442). Samples were, then, incubated overnight at 4°C in a PBS solution containing 2% FBS, anti-cardiac Troponin T (cTnT, Developmental Studies Hybridoma Bank, #RV-C2, 1:50), and anti-Perm1 (Sigma Aldrich, #HPA031712, 1:100). The samples were, then, washed three times with PBS and incubated for 60 min with a goat anti-mouse IgG2b Alexa Fluor^® 647 (Invitrogen, #A21241, 1:500), goat anti-rabbit IgG Alexa Fluor^® 488 (Invitrogen, #A11034, 1:500) and Hoechst dye (Invitrogen, #H3570, 1:500) diluted in a PBS solution. Following washing with a PBS solution three times, the samples were mounted using ProLong Gold Antifade Reagent (#P36930, Invitrogen). A three-track protocol was used in a Leica SPE confocal microscope (Leica Microsystems, Germany) equipped with a 40x lens. Images were processed using Imaris software (8.4.1 Bitplane AG, Zurich, Switzerland).

Antibodies for immunofluorescence were mouse IgG2b anti-human CD63 (clone TS63b^{35 (link)} 1/100, available upon request to E. Rubinstein: eric.rubinstein@inserm.fr) and mouse IgG1 anti-human CD9 (clone TS9^{36 (link)} 1/100) (commercially available at Diaclone or Abcam), mouse IgG1 anti-human EEA1 (BD Transduction Laboratories, clone 14/EEA1, 1/1000), mouse IgG2a anti-human RAB5 (BD Transduction Laboratories, clone 1/Rab5, 1/100), rabbit anti-human RAB7 (Cell Signaling, D95F2, 1/100), mouse IgG1 anti-human LAMP1 (Developmental Studies Hybridoma Bank, H4A3, 1/400), goat anti-mouse IgG2b Alexafluor 647 (Invitrogen, 1/300), goat anti-mouse IgG1 Alexafluor 488 (Invitrogen 1/300), goat anti-mouse IgG Alexafluor 568 (Invitrogen, 1/200), goat anti-rabbit IgG Alexafluor 568 (Invitrogen, 1/200), goat anti-rabbit IgG Alexafluor 647 (Invitrogen, 1/200).

Generation of the chicken anti‐EAAT2a (zebrafish) antibody is described elsewhere (Niklaus et al., 2017 (link)). Five dpf larvae were fixed in 4% PFA in phosphate buffered saline (PBS, pH 7.4) at room temperature for 40 min. Embryos were cryo‐protected in 30% sucrose in PBS at 4°C overnight, embedded in Tissue Freezing Medium TFMTM (Electron Microscopy Sciences), cryo‐sectioned at 14–16 μm and mounted onto Superfrost slides (Thermo Fisher Scientific). Immunohistochemistry was performed as described before (Niklaus et al., 2017 (link)). Chicken anti‐EAAT2a 1:500, mouse anti‐synaptic vesicle 2 (IgG1, 1:100, DSHB USA), mouse anti‐acetylated tubulin (IgG2b, 1:500, Sigma 7451) and mouse anti–glutamine synthetase (IgG2a, 1:200, EMD Millipore, MAB302) were used as primary antibodies. Secondary antibodies were goat anti‐chicken Alexa Fluor 488, goat anti‐mouse IgG2a Alexa Fluor 568, goat anti‐mouse IgG2b Alexa Fluor 647 and goat anti‐mouse IgG1 Alexa Fluor 647, all 1:500 (all from Invitrogen, Thermo Fisher Scientific). Slides were cover‐slipped using Mowiol (Polysciences) containing DABCO (Sigma‐Aldrich) and imaged with a TCS LSI confocal microscope (Leica Microsystems). Images were adjusted for brightness and contrast using Affinity Photo Version 1.8 and assembled in Affinity Designer Version 1.7.

The muscles from both hindlimbs were quickly removed, trimmed of excess fat and connective tissues and flash-frozen in liquid nitrogen. Muscles were cut at the mid-belly, and the distal half was mounted for tissue sectioning in a cryostat to determine muscle fiber cross-sectional area and for immunohistochemical analyses. Seven-μm thick sections were used in immunolabeling experiments to demonstrate fiber-type by probing for the major MyHC isoforms in mice skeletal muscle as previously described.^{39 (link)} Briefly, frozen sections were incubated overnight with isotype-specific anti-mouse primary antibodies for MyHC I (1:75, IgG2B, BA.D5), MyHC IIa (supernatant, IgG1, SC.71), and MyHC IIx (supernatant, IgM, 6H1) from DSHB, along with laminin (1:100, Sigma–Aldrich, St. Louis, MO, USA). Sections were then incubated with secondary antibodies (1:250, goat anti-mouse IgG2B alexa fluor 647; 1:500, IgG1 alexa fluor 488; 1:250, IgM alexa fluor 555) from Invitrogen, all diluted in PBS, along with the secondary antibody for laminin (1:150, IgG AMCA, Vector). Sections were mounted with VectaShield mounting medium (Vector), and images were captured at 20x. Fiber type distribution was quantified using Myovision as % MyHC I, IIa, IIx, and IIb (no staining).^{40 (link)} For all IHC analyses, we counted a minimum of 600 fibers for each animal and assessors were blinded to the experimental groups.

Hybridization chain reaction (HCR) and Whole Mount Immunohistochemistry were done in accordance with previous described methods [42 (link), 61 (link)]. The HCR probes transcripts were synthesized by Molecular Instruments for ret, NM_181662.2; gfra1a, NM_131730.1; oprl1, NM_205589.2; oprd1b, NM_131258.4; etv1, XM_005157634.4; elavl3, NM_131449. The following primary antibodies were used: goat polyclonal IgG anti-Choline Acetyltransferase (ChaT, Millipore Sigma, AB144P, 1:500), rabbit polyclonal IgG anti-5-HT (serotonin, Immunostar, 20080, 1:250), mouse monoclonal IgG2b anti-HuC/D (Invitrogen Thermo Fisher, A-21271, 1:250), Mouse monoclonal IgG1 anti-Phox2b (B-11, Santa Cruz Biotechnology, SC-376997, 1:250). The following secondary antibodies were used from Invitrogen: Alexa Fluor 488 donkey anti-goat IgG (A-11055, 1:600), Alexa Fluor 405 goat anti-rabbit IgG (A-48254, 1:600), Alexa Fluor 647 goat anti-mouse IgG2b (A-21242, 1:600), Alexa Fluor 594 goat anti-mouse IgG1 (A-21125, 1:600). High content semi-automated confocal imaging and processing was done as described above.