Wing discs from wandering third instar larvae were dissected in PBS, fixed in 4% formaldehyde followed by immunolabeling. Wing disc associated myoblasts were labeled using guinea pig anti-Ebd1 (1:2000; Yashi Ahmed) or rabbit anti-MEF2 (1:1000, generated here). Myoblast proliferation was monitored using rabbit anti-PH3 (1:100; Millipore). Cell death was examined using rabbit anti-Cleaved Caspase-3 (1:100; Cell Signaling). Premature differentiation was studied using Alexa-Phalloidin (Invitrogen). Localization studies were done using guinea pig anti-Htl (1:400; Christian Klambt), rabbit anti-Dof (1:200; Maria Leptin), mouse anti-GFP (1:10; DSHB), and chick anti-βgal (1:1000, ICL lab). Mouse anti- β Catenin (1:20; DSHB) was used to analyze the readout of Wg signaling. Wg ligand expression was visualized using anti-Wg (1:10; DSHB). Alexa 488 and Alexa 546 secondary antibodies (Molecular Probes) were used in this study. Samples were mounted in mounting media and imaged with an Olympus FV 3000 confocal microscope. Images were processed using Olympus Life Science software and Adobe Photoshop CC 2018.
Alexa 488 and alexa 546 secondary antibodies
Manufactured by Thermo Fisher Scientific
Alexa Fluor 488 and Alexa Fluor 546 are fluorescent secondary antibodies produced by Thermo Fisher Scientific. They are designed to bind to primary antibodies and emit fluorescent signals at specific wavelengths, enabling detection and visualization of target proteins in various biological applications.
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2 protocols using alexa 488 and alexa 546 secondary antibodies
1
Dissection and Immunolabeling of Wing Discs
2
Dissection and Immunolabeling of Wing Discs
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Wing discs from wandering third instar larvae were dissected in PBS, fixed in 4% formaldehyde followed by immunolabeling. Wing disc associated myoblasts were labeled using guinea pig anti-Ebd1 (1:2000; Yashi Ahmed) or rabbit anti-MEF2 (1:1000, generated here). Myoblast proliferation was monitored using rabbit anti-PH3 (1:100; Millipore). Cell death was examined using rabbit anti-Cleaved Caspase-3 (1:100; Cell Signaling). Premature differentiation was studied using Alexa-Phalloidin (Invitrogen). Localization studies were done using guinea pig anti-Htl (1:400; Christian Klambt), rabbit anti-Dof (1:200; Maria Leptin), mouse anti-GFP (1:10; DSHB), and chick anti-βgal (1:1000, ICL lab). Mouse anti- β Catenin (1:20; DSHB) was used to analyze the readout of Wg signaling. Wg ligand expression was visualized using anti-Wg (1:10; DSHB). Alexa 488 and Alexa 546 secondary antibodies (Molecular Probes) were used in this study. Samples were mounted in mounting media and imaged with an Olympus FV 3000 confocal microscope. Images were processed using Olympus Life Science software and Adobe Photoshop CC 2018.
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