Cy3 conjugated anti hrp

Dissected Drosophila tissues or C2C12 cells grown on coverslips were rinsed in PBS (Lonza 17-512F) and fixed in 4% paraformaldehyde (Sigma P6148) for 20 min at room temperature. Following fixation, the samples were washed four times (×10 min) in PBS and blocked with blocking buffer: 5% normal goat serum (NGS; Abcam AB7681) in PBS with 0.1% TritonX-100 (PBST). The samples were incubated with primary antibody overnight at 4 °C, washed four times (x10 min) with 0.1% PBST, incubated with secondary antibody for 2 h at room temperature followed by 0.1% PBST washes. Samples were mounted onto slides using either ProLong^® Gold Antifade mounting reagent (Invitrogen P36930) or Fluoroshield (Sigma F6057).
Primary and secondary antibodies were prepared in blocking buffer.
Primary antibodies: Cy3-conjugated anti-HRP (1:100, Jackson ImmunoResearch 123-165-021); Rabbit anti-FLAG (1:500, Sigma F7425); Mouse anti-hnRNPM 2A6 (1:100; Santa Cruz sc-20001); Rabbit anti-MATR3 (1:500; Abcam ab151714)
Secondary antibodies: Goat anti-rabbit Alexa Fluor 488 (1:1000; Invitrogen A11008); Goat anti-mouse Alexa Fluor 546 (1:1000; Invitrogen A11030)

Third instar wandering larvae were dissected, fixed, antibody stained, imaged and analyzed as described previously [49 (link), 50 (link)]. All NMJ analysis was performed double-blind. Primary antibodies used were Cy3-Conjugated anti-HRP (Goat, 1:200, Jackson ImmunoResearch Labs Cat# 123-165-021, RRID:AB_2338959) and, anti-synaptotagmin (Rabbit, 1:2000, Syt-91, RRID:AB_271399). Secondary antibodies used were anti-Rabbit IgG (H + L) Alexa Fluor 488 (1:1000, RRID:AB_2576217, goat). NMJs were imaged for quantification using an EVOS M5000 microscope. NMJ images shown were imaged using a Leica SP5 confocal microscope. NMJ bouton number and muscle surface area was quantified manually using images in ImageJ. Bouton number and length were normalized to muscle surface area. NMJ lengths were measured from stacked NMJ images using the NeuronJ plugin for ImageJ as described previously [49 (link), 50 (link)].

The antibodies used here were rabbit anti-β actin (ab8227; Abcam), mouse anti-GFP (11 814 460 001; Roche), rabbit anti-Rab7 (Tanaka and Nakamura, 2008 (link)), rabbit anti-Arl8 (Hofmann and Munro, 2006 (link)), Cy3-conjugated anti-HRP (123-165-021; Jackson ImmunoResearch, Ely, UK), rabbit anti-Syt (West et al., 2015 (link)), mouse anti-BRP [nc82; Developmental Studies Hybridoma Bank (DSHB)], mouse anti-Notch extracellular domain (C458.2H; DSHB). Information on validation of the antibodies is available from the commercial source or the indicated publication. Primary antibodies were detected by secondary antibodies conjugated with Alexa fluorochromes (Thermo Fisher Scientific) or with HRP (DAKO).
Whole flies, larvae or the indicated dissected fly tissues were lysed in equivalent amounts of SDS sample buffer and insoluble debris was removed by brief centrifugation. S2 cells growing in plates were dissolved directly in SDS sample buffer. Protein extracts were separated in 4–20% Tris-Glycine gels (Invitrogen), transferred to PVDF membranes and probed with primary and HRP-conjugated secondary antibodies, detected by chemiluminescence (ECL; GE Healthcare).