Mouse monoclonal anti discs large antibody 4f3

Brains of male flies were dissected 2 days after eclosion and mounted in phosphate-buffered saline (PBS; 137 mM NaCl, 3 mM KCl, 8 mM Na₂HPO₄, 1.5 mM KH₂PO₄, pH 7.3) for imaging native fluorescence (Figure 4C). For immunostaining (Figures 2A, 2J, and S1), dissected brains were fixed in 4% (w/v) paraformaldehyde in PBS for 20 min at room temperature, washed four times for 20 min in PBS, and permeabilized and blocked in PBS containing 0.2% (v/v) Triton X-100 and 5% (v/v) goat serum for 1 h. To label synaptic structures, the samples were first incubated with mouse monoclonal anti-discs large antibody 4F3 (Developmental Studies Hybridoma Bank, University of Iowa, 1:50) and then with Alexa Fluor 633-conjugated goat anti-mouse IgG (A-21052, Invitrogen, 1:200). Each incubation lasted for 48 h at 4°C and was followed by four 20 min washes in PBS. Stained samples were mounted in Vectashield (Vector Labs) and imaged on a Leica TCS SP5 confocal microscope equipped with an HCX PL APO 40 ×, 1.3 CS oil immersion objective (Leica). Images were processed in ImageJ.

Brains of male flies were dissected 2 days after eclosion, fixed in 4% (w/v) paraformaldehyde in phosphate-buffered saline (PBS; 137 mM NaCl, 3 mM KCl, 8 mM Na₂HPO₄, 1.5 mM KH₂PO₄, pH 7.3) for 20 min at room temperature, washed four times for 15 min in PBS containing 0.2% (v/v) Triton X-100, and mounted in Vectashield (Vector Labs) for imaging native fluoresecence. For immunostaining, fixed brains were permeabilized and blocked in PBS containing 0.2% (v/v) Triton X-100 and 5% (v/v) goat serum for 1 h. To label synaptic structures, the samples were first incubated with mouse monoclonal anti-discs large antibody 4F3 (Developmental Studies Hybridoma Bank, University of Iowa, 1:50) and then with Alexa Fluor 568-conjugated goat anti-mouse IgG (1:200) plus Alexa Fluor 633-conjugated streptavidin (1:150). Each incubation lasted for 48 h at 4°C and was followed by four 20-min washes in PBS. Stained samples were mounted in Vectashield and imaged on a Leica TCS SP5 confocal microscope equipped with an HCX PL APO 40 × /1.3 CS oil immersion objective (Leica). Images were processed in ImageJ.