Alexa fluor 647 conjugated goat anti hrp

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⁺.

Third-instar larvae were dissected in 0 Ca²⁺ HL-3 and immunostained^{71 (link)}. All genotypes were immunostained in the same tube with identical reagents, and then mounted in the same session. The following antibodies were used: mouse anti-Bruchpilot (BRP; nc82; 1:100; Developmental Studies Hybridoma Bank; DSHB); mouse anti-GluRIIA (1:100; 8B4D2; DSHB); guinea pig anti-GluRIID^{72 (link)} (1:1000); rabbit anti-DLG^{17 (link)} (1:5000); mouse anti-DLG (1:100; 4F3; DSHB); mouse anti-FK1 (1:100; Millipore 04–262); mouse anti-FK2 (1:500; BML-PW8810; Enzo Life Sciences); mouse anti-FLAG (1:500, F1804; Sigma-Aldrich); mouse anti-GFP (1:1000, 3e6; Invitrogen, Carlsbad, CA); mouse anti-pCaMKII (1:100; MA1–047; Invitrogen). Donkey anti-mouse, anti-guinea pig, and anti-rabbit Alexa Fluor 488- (715-545-150, 706-545-148, 711-545-152; Jackson Immunoresearch), DyLight 405- (715-475-150, 706-475-148, 711-475-152; Jackson Immunoresearch), and Cyanine 3 (Cy3)- (715-165-150, 706-165-148, 711-165-152; Jackson Immunoresearch) conjugated secondary antibodies were used at 1:400. Alexa Fluor 647 conjugated goat anti-HRP (123-605-021; Jackson ImmunoResearch) was used at 1:200.

Larvae were dissected as previously described (Collins et al., 2017 (link); Camuglia et al., 2018 (link)). Briefly, larvae were dissected in ice-cold 1,4-piperazinediethanesulfonic acid (PIPES) dissection buffer containing 100 mM PIPES (P6757; Sigma- Aldrich), 115 mM d-sucrose (BP220-1; Fisher Scientific), 5 mM trehalose (182550250; Acros Organics), 10 mM sodium bicarbonate (BP328-500; Fisher Scientific), 75 mM potassium chloride (P333- 500; Fisher Scientific), 4 mM magnesium chloride (M1028; Sigma- Aldrich), and 1 mM ethylene glycol tetraacetic acid (28-071-G; Fisher Scientific) and then fixed with 4% Formaldehyde in PIPES buffer (BP531-500; Fisher Scientific).
Antibodies for larva filet staining were used at the following final dilutions: rat anti- tyrosinated tubulin, 1:400 (MAB1864; Millipore), mouse anti-myospheroid (β-integrin), 1:100 (CF.6G11, Developmental Studies Hybridoma Bank). Larval NMJs were labeled with Alexa-Fluor 647-conjugated goat anti-HRP, 1:500 (123-605-021,Jackson ImmunoResearch Laboratories). We used Alexa Fluor 488- and Alexa Fluor 555- conjugated fluorescent secondary antibodies (1:400; Life Technologies), and Hoechst 33342 (1 μg/ml, ThermoFisher). Larvae were mounted in ProLong Gold (P36930; Life Technologies).
All images were acquired on a Zeiss 700 LSM using an Apochromat 40×/1.4 numerical aperture (NA) objective.