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MB Sample ID: SA239047
| Local Sample ID: | Z22 |
| Subject ID: | SU002487 |
| Subject Type: | Fish |
| Subject Species: | Danio rerio |
| Taxonomy ID: | 7955 |
| Genotype Strain: | wildtype and tango2bwh211 |
| Age Or Age Range: | 4 weeks |
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Collection:
| Collection ID: | CO002480 |
| Collection Summary: | Fish were bred and maintained using standard methods as described (Westerfield, 2000). All procedures were approved by the Brigham and Women’s Hospital Animal Care and Use Committee. tango2bwh210 and tango2bwh211 zebrafish lines were created in our laboratory by CRISPR-Cas9 approach. Zebrafish embryonic (0-2 days post fertilization) and larval stages (3-45 dpf), juvenile stage (45 dpf-3months) and adults (3 months) have been defined as described previously (Kimmel, Ballard, Kimmel, Ullmann, & Schilling, 1995). All studies presented in this work were performed on tango2bwh211 mutants obtained from heterozygous parents unless specified. Myofibers were isolated from control or tango2 larval zebrafish (45 dpf) as described previously with minor modifications (Ganassi, Zammit, & Hughes, 2021). Skinned zebrafish muscle samples were treated with collagenase for 90 minutes and triturated to release the myofibers. Myofibers were centrifuged at 1000g for 60 sec, washed and resuspended in DMEM media. Myofibers were plated on laminin coated 8 chamber permanox slides (Thermofisher Scientific) for further analysis. Fixed cells were blocked in 10% goat serum/0.3% Triton, incubated in primary antibody overnight at 4ºC, washed in PBS, incubated in secondary antibody for 1 h at room temperature (RT), washed in PBS, then mounted with Vectashield Mounting Medium (Vector Laboratories, Burlingame, CA, USA). Primary antibodies used were anti Tango2 (1:250, 27846-1-AP, Proteintech), mouse monoclonal anti sarcomeric -actinin (1:100, A7732, Millipore Sigma), mouse monoclonal anti Ryr1 (1:100, R129-100UL, Millipore Sigma), anti Tomm20 (1:100, MABT166, Millipore Sigma) and Alexa fluor 568-phalloidin (1:100, Thermo Fisher Scientific, A12380). After washing in PBS several times, samples were incubated with anti-mouse Alexa Fluor (1:100, A-11005) secondary antibody (Thermofisher Scientific). Imaging was performed using a Nikon Ti2 spinning disk confocal microscope. |
| Sample Type: | Muscle |
| Collection Method: | Muscle Dissection |
| Storage Conditions: | Described in summary |
