Alexa405

Coronal sections were prepared on a cryostat at 20 μm and immediately mounted on slides. Slides were maintained at −80°C until ready for staining. Every 10th section was Nissl stained with Cresyl Violet to identify sections corresponding to areas of interest (Franklin and Paxinos, 2013 ).
Immunostaining was done as described previously (Beaudoin et al., 2012 (link)). The following primary antibodies were used mouse anti-bassoon (1:400, ABCAM ab82958), rabbit anti-gephyrin (1:1000, ABCAM ab32206), rabbit anti-N-methyl-d-aspartate (NMDA) receptor subunit 1 (1:1000, Thermo Fisher Scientific PA3-102), and chicken anti-tyrosine hydroxylase (TH) (1:500, ABCAM ab76442). Secondary antibodies used were goat anti-mouse (1:1000 for all; Alexa568, ABCAM ab175473; Alexa488, Fisher A11029; Alexa647, Fisher A21241), goat anti-rabbit (1:1000 for all; Alexa488 ABCAM ab150077; Alexa568, Fisher A11036), and/or goat anti-chicken (1:1000, Alexa405, ABCAM ab175675). Coverslips were mounted with ProLong Gold antifade (Invitrogen).

For expression patterns, 3rd instar larval brains with RGs attached were dissected in ice-cold PBS and fixed in 3.7% formaldehyde at 4°C for 20mins. The samples were washed 4 times in PBS and mounted in 60% glycerol. Endogenous fluorescence was acquired on Olympus FV-3000 using a 20X, 40X or 60X objective, and processed used ImageJ. For samples requiring antibody staining brains were similarly processed and then subjected to permeabilisation (0.3% Triton X-100 + PBS; PBSTx) for 15 mins, 4 hr blocking in 5% normal goat serum in PBSTx at 4oC, followed by overnight incubation in primary antibody (1:1000 Chicken-GFP, Abcam: ab13970) and secondary with Alexa 488 or Alexa 594 (1:400; Abcam). For corazonin (1:1000; raised in Rabbit; Jan Veenstra, University of Bordeaux), all the above steps remained the step, except that dissected brains were fixed for 1hr at RT in 4% PFA and the secondary was anti-rabbit Alexa 405 (1:300, Abcam). Cell bodies were outlined manually and integrated density was used to calculate CTCF (Corrected Total Cell Fluorescence). For all samples, a similar area was measured for background fluorescence.

For Western blotting experiments, all primary antibodies were diluted 1:1,000 in TBS-T with 5% milk powder (MP). The following antibodies were used: α-CASK (Rb, #9497S; Cell Signaling Technologies), α-GFP (Ms, #MMS-118P; Covance), α-Veli 1/2/3 (Rb, #184 002; Synaptic Systems), α-HA (Ms, #H9658; Sigma-Aldrich), and α-Myc (Ms, #M5546, Sigma-Aldrich). HRP-coupled secondary antibodies were used in a dilution of 1:2,500 in TBS-T (Gt-α-Ms or Gt-α-Rb, #BOT-20400 or #BOT-20402; ImmunoReagents). For application in immunocytochemistry of hippocampal neurons, two primary antibodies, both prepared in 1:1,000 dilutions in 2% horse serum (HS) in PBS, were used: α-MAP2 (Ck, #ABIN 111 291; Antibodies Online) and α-vGlut1 (Rb, #135 303; Synaptic Systems). As secondary antibodies we used Alexa-405 (Gt-α-Ck, #ab175675; Abcam) and Alexa-633 (Gt-α-Rb, #A-21071; Thermo Fisher Scientific).