Immunostaining of Drosophila Larval NMJs

Third-instar larvae were dissected in modified HL3 saline and stained either with or without 0.03% Triton in PBS as described^{68 (link)}. The following primary antibodies were used: mouse anti-GluRIIΑ (8B4D2; 1:50; Developmental Studies Hybridoma Bank (DSHB)); rabbit anti-GluRIIIB^{70 (link)} (1:1000); rabbit anti-GluRIIC^{71 (link)} (1:2000); guinea pig anti-GluRIID^{70 (link)} (1:1000); rabbit anti-parvalbumin (Pa1-933; 1:1000; Thermo Fisher); mouse anti-DLG (4F3; 1:100; DSHB). The following primary antibodies were generated in this study, where the following peptides were injected into animals by Cocalico Biologicals (Stevens, PA, U.S.A): affinity purified rabbit anti-pCaMKII using the peptide C-VHRQET(p)VDCLKK (1:2000); guinea pig anti-CaMKII using the peptide C-VHRQET(p)VDCLKK (1:1000); guinea pig anti-GluRIIΑ^tail using the peptide C-SGSRRSSKEKSRSKTVS (1:2000). Alexa Fluor-647 conjugated goat anti-HRP (1:200; Jackson ImmunoResearch) and Donkey anti-mouse, -guinea pig, and -rabbit conjugated Alexa Fluor 488, Cy3, and DyLight 405 secondary antibodies (Jackson ImmunoResearch) were used at 1:400. For the BAPTA-AM treatment, dissected larvae were incubated in HL-3 saline with 0.1 or 0.3 mM BAPTA-AM (#120503; Abcam) for 30 min before fixation and then stained as described above. For the control conditions, dissected larvae were incubated in HL-3 saline in 0 of 1.8 mM Ca²⁺.