Drosophila larva transitions from larval stage L1 to larval stage L3 by intermittent molting. Prior to pupariation, L3 larvae stop feeding and migrate to a pupariation site on the side of a vial. Wandering L3 larvae were bisected and inverted in PBS, and all tissues were fixed in 4% formaldehyde/PBS for 30 min, permeabelized in 0.2% Triton X-100/PBS for 4 hours, and blocked in 3% BSA/PBS for 1 hour. All antibody stainings were performed in 3% BSA/PBS, incubation of primary and secondary antibodies were O/N. PBS was used for all rinses and washes (3× each for primary and secondary antibody incubation steps). Antibodies used were as follows: Chicken anti-RFP 1:2,000 (Rockland, 600-901-379); Goat anti-GFP 1:3,000 (Rockland, 600-101-215); Donkey anti-Goat Alexa Fluor 488 (Life Technologies, A11055); Donkey anti-Chicken Cy3 (Jackson ImmunoResearch, 703-165-155); and Donkey anti-Mouse Alexa Fluor 594 (Life Technologies, A21203). All secondary antibodies were used at 1:500. Tissues were dissected off the cuticle and were mounted in SlowFade Gold mounting medium with DAPI (Life Technologies, S36938). See Kockel et al. (2016) (link) for a detailed protocol.
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