Ab51098

Tissue was dissected in phosphate-buffered saline (PBS), fixed in 4% PFA/PBS for 30′ on ice, washed in PBS-T, incubated with primary antibody in PAXDG (PBS containing 1% BSA, 0.3% Triton X-100, 0.3% deoxycholate and 5% goat serum), followed by washing and incubation with secondary antibody in PAXDG and subsequent mounting in Vectashield with DAPI (Vector Laboratories). Primary antibodies used were: rat anti-myosin (ab51098; Abcam; 1:100) and mouse anti-pERK (M-8159; Sigma; 1:50). Polyclonal rabbit anti-CG1139 (1:200) was purified by New England Peptide Inc., MA, USA. An amino acid sequence 26–40 was selected as the epitope. The following primary antibodies were kindly gifted to us: rat anti-bnl (M. Krasnow; 1:50), guinea pig anti-Path (J. Parrish; 1:200) and rabbit anti-phospho-Drosophila S6 (pS6) (A. Teleman; 1:500). Secondary antibodies used were: Alexa Fluor 488 and 568 conjugated anti-rat antibody (A-11006 and A-11077; Thermo Fisher Scientific; 1:200), Alexa Fluor 568 conjugated anti-mouse antibody (A-11031; Thermo Fisher Scientific; 1:200), Alexa Fluor 568 conjugated anti-rabbit antibody (A-11036; Thermo Fisher Scientific; 1:200) and Alexa-568 conjugated anti-guinea pig antibody (A-11075; Thermo Fisher Scientific, 1:200). Rhodamine phalloidin (R415; Invitrogen; 1:500) was used to visualise F-actin.

For antibody staining, embryos were dechorionated, fixed and immunostained following previously published protocols (Becker et al., 2016 (link)), except that embryos were fixed for 22 min. Staging of embryos was carried out according to Campos-Ortega and Hartenstein (1997) . Early stage 17 was staged according to Pereanu et al. (2007) (link).
The following primary antibodies were used: chicken anti-Beta-Gal (1:1000; Abcam, ab9361), rabbit anti-Beta-Gal (1:1000; Cappel, 55976), mouse anti-GFP (1:250; Roche, 11814460001), rabbit anti-GFP (1:500; mTorrey Pines Biolabs, TP401), mouse anti-Fas2 1D4 (1:10), mouse anti-Cut 2B10 (1:20), mouse anti-Sxl M18 (1:10), mouse anti-Ubx FP3.38 (1:20) (all from Developmental Studies Hybridoma Bank), rat anti-Myosin (1:500; Abcam, ab51098), rabbit anti-Hb9 (1:2000; kindly provided by J. B. Skeath, Washington University in St. Louis, USA) and guinea pig anti-Ubx (1:200; kindly provided by I. Lohmann, University of Heidelberg, Germany).
As fluorescent secondary antibodies we used anti-guinea pig Dylight 405, anti-chicken Alexa Fluor 647 (both Jackson ImmunoResearch), anti-mouse Alexa Fluor 488, anti-rabbit Alexa Fluor 488, anti-mouse Alexa Fluor 568, anti-rabbit Alexa Fluor 568, anti-rat Alexa Fluor 633 (all from Thermo Fisher Scientific) at 1:500. All secondary antibodies were used according to the manufacturer's protocols.