The following Drosophila stocks were maintained at standard conditions on cornmeal medium: w1118 (Bloomington 3605), Dmef2-GAL4 [48 (link)], UAS-2xEGFP (Blomington 6874), UAS-GFP RNAi (J. Zallen, SKI). Crosses (UASxGAL4) were performed at 25°C; embryos hatched within a 2h period were selected and raised to third instar larval stage. Wandering third instar larvae were dissected and fixed in 10% formalin and labeled as previously described [16 (link)]. Muscle cells were labeled using Alexa Flour-conjugated phalloidin (Life Technologies); nuclear DNA was visualized with Hoechst 33342 (Invitogen). Anti-Lamin (DHSB, ADL 67.10) and anti-α-Tubulin (Sigma, T9026) primary antibodies, and Alexa Flour-conjugated secondary antibodies (Life Technologies) were used to label the nuclei and microtubules, respectively. Whole larvae were mounted in ProLong Gold antifade reagent (Invitrogen). VL3 and VL4 muscles in abdominal hemisegments 2-6 were imaged on a LSM 700 confocal microscope (Zeiss).
Alexa flour conjugated phalloidin
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor-conjugated phalloidin is a fluorescent dye-labeled version of the natural product phalloidin, which binds specifically to filamentous actin (F-actin) in cells. It can be used to visualize the actin cytoskeleton in fixed and permeabilized cells.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 700 confocal microscope, by Zeiss (2 mentions)
W1118, by Bloomington Drosophila Stock Center (1 mentions)
Alexa flour conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Anti α tubulin, by Merck Group (1 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
Rabbit anti homer, by Synaptic Systems (1 mentions)
Rabbitanti rab5 antibody, by Cell Signaling Technology (1 mentions)
Kt5720, by Merck Group (1 mentions)
Pkc inhibitor go 6976, by Merck Group (1 mentions)
Anti clathrin, by Abcam (1 mentions)
2 protocols using alexa flour conjugated phalloidin
1
Imaging Drosophila Larval Muscle Development
2
HA-tagged DAGLα Construct Generation
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Rat or mouse anti-HA antibodies were from Roche (Roche, Switzerland); Rabbit anti-HA, anti-V5, anti-clathrin, anti-early endosome antigen 1 (EEA1) and anti-lysosomalassociated membrane protein 1 (LAMP1) antibodies were from Abcam (Abcam, UK); Rabbit anti-Homer was from Synaptic Systems (Synaptic Systems, Goettingen, Germany); Rabbit anti-Rab5 antibody was from Cell Signaling (Massachusetts, USA).
Alexa Flour conjugated phalloidin, transferrin and secondary antibodies were from Life Technologies. PKC inhibitor Go 6976, PKA inhibitors H89 and KT 5720 were from Sigma (Sigma-Aldrich, UK). For HA-DAGLα construct, a HA peptide sequence (YPYDVPDYA) flanked by a glycine-serine-glycine-serine linker on either side was inserted into the first extracellular loop of Human DAGLα sequence in pcDNA™6.2-DEST/V5 construct (performed by GenScript, Piscataway, USA).
Alexa Flour conjugated phalloidin, transferrin and secondary antibodies were from Life Technologies. PKC inhibitor Go 6976, PKA inhibitors H89 and KT 5720 were from Sigma (Sigma-Aldrich, UK). For HA-DAGLα construct, a HA peptide sequence (YPYDVPDYA) flanked by a glycine-serine-glycine-serine linker on either side was inserted into the first extracellular loop of Human DAGLα sequence in pcDNA™6.2-DEST/V5 construct (performed by GenScript, Piscataway, USA).
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