Embryos were fixed and stained using standard protocols. Fixation was carried out using 4% paraformaldehyde diluted in PBS (phosphate-buffered saline). Specimens were incubated overnight at 4°C with primary antibodies diluted in PBT-BSA (phosphate-buffered saline containing 0.3% TritonX100% and 0.5% bovine serum albumin). The primary antibodies and concentrations used are as follows: mouse anti-Mys (βPS) integrin (1/10) (Bogaert et al., 1987 (link)); rat anti-DE-Cadherin (1/50) (Uemura et al., 1996 (link)); mouse anti-Coracle (1/100) (DSHB #C566.9); mouse anti-P-Tyr (1/400) (4G10-Upstate Biotechnology) and rabbit anti-β−Gal (1/400) (Jackson Immunoresearch). Specimens were then washed and blocked for 30 min at room temperature in PBT-BSA before a 1 h incubation with appropriate secondary antibodies (Alexa 488 and Alexa 546—fluorescent-conjugated antibodies from Molecular Probes and horseradish peroxidase (HRP) conjugated goat anti-mouse and goat anti-rabbit from Jackson Immunoresearch diluted 1:200 in PBT-BSA at room temperature.
Specimens were mounted in 50% Glycerol or Vectashield (Vector Laboratories) and examined under a Zeiss Axioplan II, a Radiance 2000 Bio-Rad and a Zeiss LSM700 confocal microscope. Superesolution was achieved in a Zeiss AiryScan LSM880 confocal microscope with a ×100 objective.
Specimens were mounted in 50% Glycerol or Vectashield (Vector Laboratories) and examined under a Zeiss Axioplan II, a Radiance 2000 Bio-Rad and a Zeiss LSM700 confocal microscope. Superesolution was achieved in a Zeiss AiryScan LSM880 confocal microscope with a ×100 objective.