Antibody staining was performed according to [91 (link)]. Primary mouse anti-SSp40 (1:1000) [20 (link)] and Cy3 AffiniPure donkey anti-mouse secondary (1:200) (Jackson Immuno Research) were used to detect Lh EVs. Mouse anti-Antennapedia (1:10; Developmental Studies Hybridoma Bank 8C11, [92 ] and macrophage-specific mouse anti-P1 (1:20; I. Ando [93 (link)]) were similarly detected. Nuclear dye (Hoechst 33258, Invitrogen, 1:500) and Rhodamine or Alexa Fluor 488-tagged Phalloidin (Invitrogen) were used for counterstaining cells. For mitotic index, rabbit anti-phospho-histone3 (1:200 Molecular Probes)-positive hemocytes were scored in randomly selected 1000 μm2 areas of imaged lobes.
Samples were mounted in VectaShield (Vector Laboratories). Lamellocytes were visualized by (a) high F-actin staining signal, (b) integrin-β (1:200, Developmental Studies Hybridoma Bank CF.6G11 [94 (link)]) expression, or (c) msnf9-GFP expression [88 (link)]. Representative results from twelve or more dissections from at least three independent experiments are presented, unless specified otherwise.
Samples were mounted in VectaShield (Vector Laboratories). Lamellocytes were visualized by (a) high F-actin staining signal, (b) integrin-β (1:200, Developmental Studies Hybridoma Bank CF.6G11 [94 (link)]) expression, or (c) msnf9-GFP expression [88 (link)]. Representative results from twelve or more dissections from at least three independent experiments are presented, unless specified otherwise.