Ab7681

Wandering 3rd instar, larvae from the F1 generation were rinsed in ice-cold phosphate-buffered saline (Lonza; 17-512 F) and dissected along the dorsal midline. Muscles and NMJs were exposed by removing all tissues except the brain and nerves. The dissected larval pellet was fixed in 4% paraformaldehyde in PBS for 20 min at room temperature, washed with PBS, and then blocked with 5% normal goat serum (NGS) (Abcam; AB7681) in 0.1% PBST (0.1% Triton X-100 in PBS). Larval pellets were then probed with primary antibodies overnight at 4°C, washed several times with 0.1% PBST incubated with secondary antibodies for 2 h at room temperature, and then washed with 0.1% PBST. Both primary and secondary antibody solutions were prepared in 5% NGS in 0.1% PBST. Samples were mounted onto slides using ProLong Gold Antifade mounting reagent (Invitrogen; P36930). Confocal images were acquired using a Fluoview FV1000 confocal laser scanning system equipped with a IX81 microscope (Olympus) and a × 60 oil objective. For the analyses, the innervating muscle 4 of the NMJs on segments A2–A5 were imaged, and the synaptic bouton were quantified. Boutons included in a chain of two or more are considered mature. Satellite boutons are not in a chain and sprout off of a mature bouton or branch. Statistical analyses were performed with Prism 6 (GraphPad Software).

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)