Rabbit anti pkcζ

Brains and egg chambers were fixed for 20 min in 4% formaldehyde (Sigma-Aldrich) in PBS. Primary antibodies were incubated in PBST (PBS containing 0.1% Triton X-100) overnight. Primary antibodies used were as follows: rabbit anti-PKCζ (Santa Cruz, sc-17781, 1:1000), guinea pig anti-Numb (1:1000, provided by J. Skeath, Department of Genetics, Washington University in St. Louis, USA; O'Connor-Giles and Skeath, 2003 (link)), rabbit anti-Miranda (1:1000, provided by C. Gonzalez, Institute for Research in Biomedicine, Barcelona, Spain; Mollinari et al., 2002 (link)), rabbit anti P-S980-Bazooka,(1:1000, provided by A. Wodarz; Krahn et al., 2009 (link)), guinea pig anti-Par6 (1:500, provided A. Wodarz; Kim et al., 2009 (link)), mouse anti-Dlg (Developmental Studies Hybridoma Bank, 4F1, 1:500). Secondary antibodies used were (Life Technologies, all used at 1:2000): goat anti-rabbit Alexa 488 (A11034), goat anti-guinea pig Alexa 647 (A21450), goat anti-mouse Alexa 647 (A21325), goat anti-mouse Alexa 488 (A11029), donkey anti-rabbit Alexa 594 (A21207). Images were taken using a Leica-SP8 confocal microscope with a 63× water objective (NA 1.2).

To analyze the UAS-RNAi lines of the screen, late L3 larval brains were dissected, mounted without fixation, and analyzed under a Carl Zeiss microscope (Axio Imager.A1), EC Plan-Neofluar 20× objective (Figure 4, Figure 5 and Figure 6a) and an AxioCam Hrc Carl Zeiss camera. Images were assembled using Adobe Photoshop CS6.
To perform the immunofluorescence, L3 larval brains were dissected in PBS and fixed with 4% PFA in PBT (PBS and Triton X-100 0.1%) for 20 min at room temperature with gentle rocking. Fixed brains were washed 3 times for 15 min with PBT (PBS and Triton X-100 0.3%) and then incubated in PBT-BSA for at least 1h before incubation with the corresponding primary antibody/antibodies. The following primary antibodies were used in this study: guinea pig anti-Dpn (1:2,000; [42 (link)]), rabbit anti-Ase (1:100; [42 (link)]), goat anti-Numb (1:200; Santa Cruz Biotechnology, sc-23579), and rabbit anti-PKCζ (1:100; Santa Cruz Biotechnology, sc-216). Fluorescence images corresponding to Figure 1 and Figure 2a,b were recorded using an Inverted Leica laser-scanning spectral confocal microscope TCS SP2. Fluorescence images in Figure 2c and Figure 6b were recorded using a Super-resolution Inverted Confocal Microscope Zeiss LSM 880-Airyscan Elyra PS.1 (Figure 2c) or an Inverted Confocal Microscope Olympus FV1200 (Figure 6b), respectively.

Ovary dissection was carried out in phosphate-buffered saline (PBS) and then fixed in Devitellinizing buffer (7% formaldehyde) and heptane (Sigma) mixture (1:6) for 10min. After washes in PBS, ovaries were incubated in blocking solution (PBT, 10% goat serum) for 30min and then stained overnight at 4°C. Primary antibodies and their concentrations were as follows: mouse anti-Arm (1:50; N27A1; Developmental Studies Hybridoma Bank (DSHB)), rabbit anti-PKCζ (1:200; C-20; Santa Cruz), mouse anti-Dlg (1:50; 4F3; DSHB), mouse anti-Crb (1:10; Cq4; DSHB), rat anti-Ncad (1:20; DN-Ex #8; DSHB). Methanol treatment was used after fixation for anti-Crb staining. After washes in PBT, ovaries were incubated with secondary antibodies (1:250, Jackson ImmunoResearch) for 2 hours at room temperature. F-actin was labeled by Rhodamine phalloidin (1:150, Sigma). DAPI (0.05 mg/mL, Sigma) was used to stain nuclei. Confocal images were obtained using a Leica TCS SP5 II with a HyD detector or an Olympus FV1000 confocal microscope with a GaAsP detector. High resolution confocal images were obtained using a Zeiss LSM 880 with Airyscan super-resolution mode. Three-dimensional confocal reconstruction were performed by Imaris software (Bitplane).