Immunocytochemistry imaging

Wandering third instars were dissected in physiological saline (see above) and fixed in 4% paraformaldehyde (EMS, 15714) diluted in PBS (Corning, 46–013-CM) for 10 minutes at RT. Preparations were then washed and permeabilized in PBS containing 0.2% Triton X-100 and 1% bovine serum albumin (BSA; 3X, 10 minutes), followed by blocking for 30 minutes at RT in the same solution. Preparations were incubated with primary antibodies overnight at 4°C. Primary antibodies used included rabbit anti-pERK1/ERK2 (Thr185, Tyr187) polyclonal antibody (Thermo Fisher, 44-680G, 1:100), goat Cy3-conjugated anti-HRP (Jackson ImmunoResearch, 123–165–021, 1:200), and goat 488-conjugated anti-HRP (Jackson ImmunoResearch, 123–545–021, 1:200). Preparations were washed (3X, 10 minutes) and then incubated with secondary antibodies for 2 hours at RT. Secondary antibodies used included: donkey 488 anti-rabbit (Invitrogen, A21206) and donkey 555 anti-rabbit (Invitrogen, A31572). Preparations were washed (3X, 10 minutes) and then mounted in Fluoromount G (Electron Microscopy Sciences) onto 25 × 75 × 1 mm slides (Fisher Scientific, 12–544–2) with a 22 × 22–1 coverslip (Thermo Fisher Scientific, 12–542-B) sealed with clear nail polish (Sally Hansen). All NMJ imaging was performed using a Zeiss LSM 510 META laser-scanning confocal microscope, with images projected in Zen (Zeiss) and analyzed using ImageJ (NIH open source). All NMJ intensity measurements were made with HRP signal-delineated z-stack areas of maximum projection using ImageJ threshold and wand-tracing tools.