Wandering third-instar larvae for various genotypes were dissected and fixed in Bouin’s fix for 15 minutes and processed simultaneously as previously described11 (link). Confocal images of all genotypes of larvae belonging to the same experimental group were acquired using same settings with a Zeiss LSM710 confocal microscope.
The following primary antibodies were used: FITC-conjugated anti-HRP (1:250, Jackson ImmunoResearch laboratories), Rabbit anti-Syt31 (link) (1:1000, H. Bellen, Baylor College of Medicine), mouse monoclonal anti-Dlg (1:1500, 4F3), anti-Brp (1:250, NC82), anti-DCSP2 (1:250, 6D6) and anti-Futsch (1:1000, 22C10) obtained from Developmental Studies Hybridoma Bank (DSHB), University of Iowa. Anti-Hrp conjugated to Alexa 488 or 568 were used at 1:250. Secondary antibodies conjugated to Alexa 488, 568, and 647 (Invitrogen-Molecular Probes) were used at 1:200.
The following primary antibodies were used: FITC-conjugated anti-HRP (1:250, Jackson ImmunoResearch laboratories), Rabbit anti-Syt31 (link) (1:1000, H. Bellen, Baylor College of Medicine), mouse monoclonal anti-Dlg (1:1500, 4F3), anti-Brp (1:250, NC82), anti-DCSP2 (1:250, 6D6) and anti-Futsch (1:1000, 22C10) obtained from Developmental Studies Hybridoma Bank (DSHB), University of Iowa. Anti-Hrp conjugated to Alexa 488 or 568 were used at 1:250. Secondary antibodies conjugated to Alexa 488, 568, and 647 (Invitrogen-Molecular Probes) were used at 1:200.
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