0.1% SDS, 10 mM beta-mercaptoethanol, 10 mg/mL PMSF, 5 mg/mL aprotinin, 0.1 mg/mL benzamidine, 1 mg/mL pepstatin A, 1 mg/mL leupeptin, 100 mM sodium orthovanadate, 15 mL/ mL Triton X-100H, in Phosphate Buffered Saline (PBS) pH 7.4) on ice for 45 min and homogenized by two 15-sec pulses. Lysates were centrifuged at 14,000 g for 10 min at 4uC and supernatants were collected. Protein concentration was determined using the Bradford assay (Bio-Rad, Hercules, CA). Proteins (20 mg/lane) were separated by SDS-PAGE alongside Kaleidoscope pre-stained standards on 4–15% Tris-HCl gradient gels (Bio-Rad; 100 V, 120 min) and transferred onto PVDF membranes using a Transblot SD semi-dry electrophoretic transfer cell (Bio-Rad) following manufacturer’s instructions. Membranes were washed twice with TBS-T [0.1% Tween-20 in Tris buffered saline (TBS)], blocked in blocking buffer (3% non-fat dry milk in TBS-T) for 60 min at RT, and incubated with anti- Pax7 antibody (1:50; Developmental Hybridoma Bank, Iowa City, IA) diluted in 5% BSA in TBS-T overnight at 4uC. After PBST washes, membranes were incubated with an HRP-conjugated secondary antibody for 1 hr. The BioRAD amplification module was used according to manufacturer’s instructions. Colorimetric detection via BioRAD’s Opti-4CNTM Substrate Kit was per- formed according to manufacturer’s instructions to visualize antigen-antibody complexes. In vitro Translation of S. macrururs Pax7 cDNA To further verify antibody specificity to S. macrurus Pax7 protein, a 1,435 bp Pax7 fragment from muscle was ligated into the pGEMTeasy vector, (Promega), and subcloned. Following se- quence verification, the insert from muscle cDNA was translated in vitro using the TNTH T7 Coupled Reticulocyte Lysate System (Promega) following manufacturer’s instructions. Translation product was then separated by SDS PAGE (product diluted 46 in 26 SDS load buffer & 20 mL, 25 mL, and 30 mL were loaded), transferred to PVDF, and probed with the Pax7 antibody as described above. Electron Microscopy Normal adult tails (n = 2) were examined under a transmission microscope (TEM). After surgical removal, blastemas and tails were fixed in 4% paraformaldehyde/2.5% gluteraldehyde over- night, transferred to sodium phosphate buffer (pH = 7.2), post- fixed in osmium (2% osmium in sodium phosphate buffer) for 1 hour, dehydrated in an alcohol series and propylene oxide, and embedded in Spurrs’ plastic resin (Polysciences, Warrington, PA). Ultrathin (90-nm) tissue cross sections were cut with a diamond knife, stained with uranyl acetate and lead citrate, and examined with the Hitachi HU11-E TEM (Cell Research Institute of the University of Texas at Austin). Imunofluorescence Serial transverse and longitudinal cryosections (14–30 mm thick) were mounted on glass slides (Superfrost Plus, Fisher, Pittsburgh, PA) and air-dried at room temperature. Sections were surrounded with a hydrophobic barrier by using an ImmEdge pen (Vector Laboratories, Ingold, CA) to retain incubating solutions on the tissue. Sections were post-fixed on slides by immersion in 4% buffered formaldehyde (pH 7.4, 20 min, 37uC) and rinsed twice in 0.1 M PBS (pH 7.4). Slides were immersed in permeabilization solution (0.5% TritonH X-100 in PBS for 30 min.) and subsequently incubated in 1% blocking reagent (TSA kit, Invitrogen) for 1 hr at room temperature. Mouse monoclonal antibodies against Pax7 (1:20), sarcomeric myosin heavy chain isoforms (MF20, 1:100), and laminin (1:100) were diluted in 1% Myogenic Stem Cell-Dependent Regeneration in Fish blocking solution and applied to separate slides for 15–20 h at room temperature. Antibodies against Pax7 and MF20 were obtained from the Developmental Hybridoma Bank (Iowa City, IA) and the anti-laminin antibody was obtained from Sigma (St. Louis, MO). Tissue sections were incubated in 1% blocking solution without primary antibodies to assess nonspecific labeling by secondary antibodies. Sections were rinsed in PBS and incubated in horseradish peroxidase-conjugated goat anti-mouse IgG (TSA kit, diluted 1:100 in 1% blocking solution) for 1 h. Tissue sections were rinsed in PBS, and incubated in Alexa Fluor 488 or 546 tyramide working solution (TSA kit, 10 min., diluted 1:1000 in 0.0015% H2O2). Sections were rinsed in PBS, counterstained with propidium iodide (Sigma; 1:1000, 1 min) or SYTOX Green nucleic acid stain (Invitrogen, 1:1000, 1 min), rinsed in deionized water, and cover-slipped with Vectashield Hard Set mounting media (Vector) or Gelmount (Biomeda, Foster City, CA). BrdU Immunolabeling Fish were given intraperitoneal injections of BrdU (Sigma) at a dose of 100 mg/kg. Fish were given two BrdU injections: immediately after tail amputation and 24 h after tail amputation. In these fish, the distal-most 50 mm portion of the tail stump was removed 7 or 14 days post amputation. For each group, non- amputated control fish were injected (n = 2) to determine the level of cell replication in the absence of tail amputation. The fates of BrdU-labeled cells in regeneration blastema were assessed 1 (n = 5) and 2 (n = 5) weeks after tail amputation. Serial transverse and longitudinal cryosections (20–30 mm thick) were mounted on glass slides, and air-dried at room temperature. Slides were immersed in 0.1 M PBS for 5 min followed by incubation in HCl solution (1.5 mL HCl/23.5 mL ddH2O) for 30 min. at 37uC. Slides were rinsed in PBS, incubated in.1 M sodium borate (pH = 7–8) for 10 min at room temperature, and then immersed in 1% blocking solution (Invitrogen) for 30 min prior to incubation in mouse anti- BrdU antibody (BD biosciences, San Jose, CA; 1:20) for 15–20 h at room temperature. Slides were rinsed in PBS and incubated in fluorescein-conjugated secondary antibody (Alexa Fluor 488 or 546; dilution, 1:200; Molecular Probes, Eugene, OR) for 1 hr. Sections were again rinsed in PBS, counterstained in propidium iodide or SYTOX, rinsed in deionized water, and cover-slipped with Vectashield Hard Set mounting media (Vector) or Gelmount (Biomeda). Intracellular Dye Injections To test the incidence of cell dedifferentiation during regener- ation in S. macrurus, fluorescent lineage tracer lysinated tetra- methylrhodamine (Fluororuby; 10,000 MW; Molecular Probes, Inc.) was pressure-injected with glass micropipettes and a picospritzer into individual electrocytes or muscle fibers (n = 10– 15/fish) of a total of 22 fish under a Zeiss Axiovert 100 M fluorescent microscope. A flap of skin was reflected to allow visualization and access to individual cell types. Fluororuby exhibits low toxicity and is retained in cells for periods of up to 3–4 weeks with little incidence of diffusion [34;35]. Tails were transected immediately after muscle fibers and electrocytes were microinjected with the dye and cell filling with the dye verified under a fluorescent scope. At 1-, 2-, 3-, 4-, 5- and 6-days post- amputation, proximal stumps were processed for cryosectioning and immunolabeling with Pax7 and MF20 antibodies using methods described above. PLoS ONE | www.plosone.org 4 May 2012 | Volume 7 | Issue 5 | e36819