Slowfade medium

Muscles were fixed in 4% PFA for 90 min, washed (0.1 M glycine in PBS) for 30 min, permeabilized (1% [v/v] Triton X-100 in PBS) for 90 min, and incubated in 5% (w/v) bovine serum albumin with 1% Triton X-100 in PBS for 3 h. Samples were incubated overnight at 4 °C, with the following primary antibodies: rabbit polyclonal anti-SMN H195 (sc-15320, SCBT), mouse monoclonal anti-NF-M (sc-51683, SCBT), and goat polyclonal anti-MAP1B (sc-8970, SCBT). The next day, muscles were incubated in PBS containing 0.05% Triton X-100 for 1 h, exposed to the appropriate secondary antibodies for 1 h (Alexa Fluor 647-conjugated donkey anti-goat or donkey anti-rabbit (Invitrogen, Madrid, Spain), Alexa Fluor 647-conjugated donkey anti-chicken (Jackson 703-605-155), Alexa Fluor 594-conjugated donkey anti-rabbit (Invitrogen, Madrid, Spain), CF488-conjugated donkey anti-mouse (Biotium, Madrid, Spain), plus 10 ng/mL bugarotoxin-rhodamine- (BTX-rho), and bathed again with 0.05% Triton X-100 for 90 min. Finally, muscles were mounted with Slowfade medium (Invitrogen, Madrid, Spain)).

Third instar larvae were dissected in PBS and digestive tracks, salivary glands, and central nervous system were fixed in PBS with 4% formaldehyde. Tissues were then rinsed in PBS with 0.1% triton and incubated with polyclonal anti-pebble guinea pig antibodies (GP14)^{19 (link)} diluted at 1/1000 over night at 4 °C. After four washes in PBS with 0.1% triton, the preparations were incubated with Cy2-coupled anti-guinea antibodies (P0141, DAKO) diluted at 1/500. After four washes in PBS with 0.1% triton and a rinse in PBS, the tissues were mounted in Slowfade medium containing DAPI (Invitrogen) to visualize the DNA.

Cultured motoneurons were fixed with 4% paraformaldehyde for 20 min (10 min on ice, 10 min at RT), permeabilized with 1% (v/v) Triton X-100 in PBS washing three times for 5 min each time. Cultures were incubated with the following primary antibodies: anti-bassoon (mouse monoclonal, 1:250, Enzo); anti-SMN (rabbit polyclonal, 1:250, Santa Cruz); anti-vGLUT2 (rabbit policlonal, 1:250, Synaptic Systems); anti-PSD-95 (mouse monoclonal, 1:250, Millipore); anti v-AChT (rabbit polyclonal, 1:250; Synaptic Systems) diluted in 5% (w/v) BSA, 1% Triton X-100 in PBS for 1 h. After washing three times for 5 min with 1% Triton X-100 in PBS, cells were incubated for 1 h with the secondary antibody (Alexa 594 or Alexa 647-conjugated donkey anti-rabbit or anti-mouse, Invitrogen) in PBS 1X containing 0.05% Triton X-100 (1:500) and washed with PBS three times for 5 min. Finally, cells were mounted with slowfade medium (Invitrogen) on microscope slides.

Tissues were embedded directly in an optimal cutting temperature compound without fixation or placement in 10% formalin overnight, and then embedded in paraffin and sectioned at 5 μm. Sections were de-waxed in xylene and rehydrated in successive ethanol baths. For immunohistochemistry (IHC), the MOM kit was used (Vector). H&E and CD34 staining were performed in Translational Pathology Shared Resources (Vanderbilt University, Nashville, TN, USA). For immunofluorescence (IF) staining, primary and secondary antibodies were diluted in 12% BSA, and then mounted in DAPI that contained a SlowFade medium (Invitrogen). Antibodies used for staining were NG2 (1:200; Abcam), CD31 (1:200; BD Biosciences), 5-bromo-2’-deoxyuridine (BrdU) (BD Biosciences). Quantification of staining was performed using ImageJ software (National Institutes of Health, Bethesda, MD, USA) in accordance with the recommended guidelines. H&E and IHC sections were photographed using the OLYMPUS BX41 microscope and OLYMPUS DP2-BSW software. Slides for H&E and CD34 staining of lungs were scanned using the Leica SCN400 slide scanner with 20 × objective. Slides were photographed using a ZEISS Axioplan 2 microscope, and then numbered using MetaMorph software.

Cryosections (10 μm) of lung parenchyma frozen biopsies were obtained using a cryostat (Leica CM3050S, Nanterre, France). Sections were fixed in methanol/acetone (1:1) at -20°C for 20 min and stained with sheep IgG anti-FITC IgG in order to amplify the CFSE signal, followed by donkey anti-sheep IgG-A594 and with matched sheep IgG controls (S1 Table). Sections were stained with DAPI and mounted in SlowFade medium (Invitrogen). The sections were scanned at a x 20 magnification with the Pannoramic SCAN II, v3.0.2.