Immunohistochemical Analysis of Insect Tissues

Larval brains and ovaries from well-fattened females were dissected and fixed as previously described (29 (link)). S2R+ culture cells were fixed and mounted as described (29 (link)). In short, samples were fixed in Grace's Insect Medium (modified) (BioWhittaker, Lonza, Cologne, Germany) containing 4% paraformaldehyde and 20 mM formic acid (Sigma-Aldrich Corp. St. Louis, MO, USA). Antibody staining was performed in antibody wash buffer (1× PBS:0.1% Triton X-100:1% BSA). The following antibodies were used: rabbit anti-GFP (1:2000, Torrey Pines Biolabs, Secaucus, NJ, USA), mouse anti-myc (1:1000, Sigma-Aldrich Corp. St. Louis, MO, USA), anti-FLAG (1:1000, Sigma-Aldrich Corp. St. Louis, MO, USA), mouse anti-ATP synthase (1:1000, CVA, Mitosciences) and Phalloidin (1:250, Invitrogen). The following secondary antibodies were used: goat anti-mouse IgG2_b Alexa 488, goat anti-mouse IgG2_b Alexa 568, goat anti-mouse IgG₁ Alexa 488 and goat anti-rabbit Alexa 488 (Molecular Probes, Lifetechnologies, Grand Island, NY, USA). Images were collected using Zeiss 710 and Zeiss 700 confocal microscopes and 63× Plan Apo NA 1.4 lens.