Chat4b1

Third instar larvae were dissected, fixed, and immunostained as described [74] (link). Briefly, larvae were dissected in dissection buffer (2X stock contains 128 mM NaCl, 4 mM MgCl₂, 2 mM KCl, 5 mM HEPES, and 36 mM sucrose, pH 7.2). Dissected larvae were fixed in 4% formaldehyde and incubated with primary antibodies against either cysteine string protein (CSP, 1∶10, Developmental Studies Hybridoma Bank), Syntaxin (8C3, 1∶5, Developmental Studies Hybridoma Bank), Highwire (6H4, 1∶5, Developmental Studies Hybridoma Bank), Futsch (22C10, 1∶5, Developmental Studies Hybridoma Bank), Discs Large (4F3, 1∶5, Developmental Studies Hybridoma Bank), Choline Acetyltransferase (ChAT4B1, 1∶5, Developmental Studies Hybridoma Bank) or kinesin heavy chain antibody (SUK4, 1∶5, Developmental Studies Hybridoma Bank) overnight. Larvae were incubated in HRP-TR and secondary antibody (Alexa anti-rabbit 488, 1∶100, Invitrogen) for 2 hrs at room temperature, mounted using Vectashield mounting medium (Vector Labs) and imaged using a Nikon TE-2000E inverted microscope at 60X.

Two day old cultures were fixed in 8% paraformaldehyde for 30 min at 4°C. After three washes in 1X PBT cultures were incubated in primary antibody against either Bruchpilot (nc38, 1∶5, Developmental Studies Hybridoma Bank), GluRN2A (1∶100, provided by Dr. DiAntonio [42] (link)), cysteine string protein (CSP, 1∶10, Developmental Studies Hybridoma Bank), Syntaxin (8C3, 1∶5, Developmental Studies Hybridoma Bank), Highwire (6H4, 1∶5, Developmental Studies Hybridoma Bank), Futsch (22C10, 1∶5, Developmental Studies Hybridoma Bank), Discs Large (4F3, 1∶5, Developmental Studies Hybridoma Bank), Choline Acetyltransferase (ChAT4B1, 1∶5, Developmental Studies Hybridoma Bank) or kinesin heavy chain antibody (SUK4, 1∶5, Developmental Studies Hybridoma Bank). Cultures were incubated in Alexa 488 anti-mouse (1∶100, Invitrogen), HRP-TR (1∶50, Jackson Immunoresearch Laboratories), and/or DAPI (1∶20, Invitrogen) for 2 hrs at room temperature. Cultures were mounted using Vectashield mounting medium (Vector Labs) and imaged using a Nikon TE 2000-E inverted microscope at 60X and 100X.

Brains were dissected and fixed as described by Pfeiffer et al. [73 (link)]. Brains were reacted with ChAT4B1 and DN-Ex #8 (both from Developmental Studies Hybridoma Bank) to detect ChAT and CadN), followed by incubation with Cy3 conjugated anti-mouse and Cy5 conjugated anti-rat F(ab')₂ fragments (both from Jackson ImmunoResearch). After the final wash, they were rinsed 3 × 5 min in PBS with 0.1% Triton X-100 at room temp, incubated for 2 hr with 1:500 α-Bungarotoxin, Alexa Fluor 488 conjugate (ThermoFisher Scientific) in PBS 0.1% TX-100, rinsed 3 × 5min with PBS 0.1% TX-100, mounted in SlowFade Diamond Antifade Mountant (ThermoFisher Scientific) and visualized immediately. All experiments were carried out blind.