Sab4502380

Whole mount retinas at 40% after puparium formation (APF) were prepared as previously described (Walther and Pichaud, 2006 (link)). The following antibodies were used: rabbit anti-PKCζ (1:600, SAB4502380, Sigma), mouse anti-Arm (1:200, N27-A1, Developmental Studies Hybridoma Bank), rat anti-Baz, (1:1000, a gift from Andreas Wodarz, University of Cologne, Germany), rabbit anti-Cno, (1:200, a gift from Linda Van Aelst, Cold Spring Harbor Laboratory, New York, USA Boettner et al., 2003 (link)), rabbit anti-Baz (1:2000), rat anti-Crb (1:200), guinea pig anti-Mbt (1:200) (Walther et al., 2016 (link)), with the appropriate combination of mouse, guinea pig, rabbit and rat secondary antibodies conjugated to Dy405, Alexa Fluor 488, Cy3 or Cy5 as appropriate at 1:200 each (Jackson ImmunoResearch) or TRITC-conjugated Phalloidin (P1951, Sigma) at 2 μg/ml. Retinas were mounted in VectaShield™ with or without DAPI as appropriate, and imaging was performed by using a Leica SP5 confocal microscope. Images were edited with ImageJ and Adobe Photoshop 7.0.

All procedures (TEM and Immunofluoresence) were performed as previously described [14 (link), 15 (link)]. The primary antibodies used in this study were: mouse anti-elav (9F8A9, 1:200, Developmental Studies Hybridoma Bank); mouse anti-Crumbs (Cq4, 1:100, Developmental Studies Hybridoma Bank), rabbit anti-aPKC ζ (zeta) (1:200, SAB4502380 Sigma), mouse anti-HA (6E2, 1:500, Cell Signaling), mouse anti-myc (9B11, 1:500 Cell Signaling), rat anti-Crumbs (1:500) [73 (link)], mouse anti-armadillo (N2 7A1, 1:100, Developmental Studies Hybridoma Bank). The C-terminal peptide KVNKLISRFEGGRPRLC (produced in the laboratory of Dr. Charles Zuker) was used as the antigen in rabbits, and the resulting antibody was used at a concentration of 1:200. Fluorescent conjugated secondary antibodies were obtained from either Jackson ImmunoResearch Laboratories or Life Technologies. Rhodamine or Alexa Fluor 647 conjugated phalloidin (1:200, Life Technologies) were utilized for the detection of F-Actin. Confocal images were captured on a Leica TCS SP5. TEM imaging was conducted with a JOEL 1010 and JOEL 1400. All images were processed in Adobe Photoshop.