Mouse anti fmi

Drosophila pupal wings were prepared for imaging as previously described (Axelrod, 2001 (link)). Primary antibodies were as follows: mouse anti-Fmi (#74, Developmental Studies Hybridoma Bank), mouse anti-Arm (Developmental Studies Hybridoma Bank), rabbit anti-alpha Tubulin (Abcam, Cambridge, UK), rat anti-tyrosinated Tubulin (Abcam, Cambridge, UK), rat anti-Ds and rat anti-Ft (Yang et al., 2002 (link)). Images were acquired on a Leica TCS SP5 AOBS confocal microscope using a 63x objective and processed with LAS AF (Leica).

The head and the bottom of the pupae was dissected in PBS and quickly transferred in PFA 4% at room temperature for one hour. Dissected pupae were transferred into PBS-TN (PBS-0.3% Triton-20% NGS), the pupal case was removed and finally the wing was extracted. Washes were done in PBS-TN. Primary antibodies were incubated in PBS-TN, overnight at 4 °C. Secondary antibodies were incubated in PBS-TN for 1 hour at room temperature.
For Crb localization at SAR antibody staining in pupal wings were performed as previously described^{39 (link),68 (link)} by using PBS 0.01% Triton X-100. Primary antibodies were: mouse anti-Fmi [1:20], mouse 4F3 anti-Dlg and rat anti-DE-Cad2 [1:100] from Developmental Studies Hybridoma Bank (DSHB, University of Iowa, USA); rat anti-Crumbs2-8^{7 (link)}, rabbit anti-DPatj^{69 (link)}, rat anti-Sdt^{62 (link)}, rabbit anti-Moesin^{70 (link)}, rat anti-Yurt^{15 (link)}; anti-Myosin II^{71 (link)}. Secondary antibodies and Rhodamine-conjugated phalloidin were from Molecular Probes and Jackson ImmunoResearch Laboratories. Confocal images were acquired at 40x, 63x and 100x magnification on a LSM 510 Zeiss Confocal Microscope. Confocal sections were spaced 0.5 μm apart.

The following antibodies were used:

mouse anti-Fmi (1:10; Developmental Studies Hybridoma Bank),

mouse anti-Fascin (1:10; Developmental Studies Hybridoma Bank),

mouse anti-Scar (1:50; Developmental Studies Hybridoma Bank),

rabbit anti-Dia (1:1,000, donated by P Adler),

mouse anti-Chic (1:10; Developmental Studies Hybridoma Bank),

rabbit anti-PatJ (1:500), mouse anti-HA (1:50; BioLegend),

mouse anti-α-actin (1:1,000; Sigma-Aldrich).

All fluorophore-conjugated secondary antibodies were used at 1:200 and obtained from Jackson ImmunoResearch Laboratories, Inc. Rhodamine-phalloidin was from Invitrogen and used at 1:400.