Tcl 119 100ml

Larvae were dissected in 1X PBS, followed by fixing in 4% paraformaldehyde (Himedia—TCL-119 - 100ml) for 30 minutes at room temperature and three washes in 1X PBS with 0.2% TritonX-100 (Himedia—MB031, 1X PBST). Primary antibodies were incubated overnight at 4°C. Followed by blocking in 5% normal goat serum (Himedia—RM10701) for 1h at room temperature and then secondary antibody incubation followed by washing and dissection. Samples were mounted in Vectashield (VectorLabs—H100) and imaged under 40X or 63X oil immersion Leica Stellaris 5 or Olympus FV3000 confocal microscopes. Images were processed using Fiji. All antibody dilutions and the blocking solution were made in 1X PBST; details of antibodies and their dilutions used are listed in Table 2.

Cells were fixed in 4% paraformaldehyde (TCL119-100ML, Himedia, Mumbai, India), permeabilized with 0.2% TritonX-100 (T9284, Sigma-Aldrich, St. Louis, Missouri, USA), and blocked with 1% bovine serum albumin (BSA) (A7906, Sigma-Aldrich) in phosphate buffered saline (PBS) (Gibco, Grand Island, NY, USA)for 1 h at room temperature. The cells were then incubated with primary antibody at 4 °C overnight, followed by secondary antibody for 1 h at room temperature. The nuclei were stained with 4′,6-diamidino-2-fenylindool (DAPI; Sigma-Aldrich). Images were taken by confocal microscopy (Leica Microsystems, Wetzlar, Germany).