Flies were kept at standard laboratory conditions and raised on corn-meal medium at 25°C, 12:12 h light:dark cycle. We used the following lines: w
, canton S (CS), GH146- Gal4, pdf- Gal4, MB247- Gal4, UAS-CD8::GFP, pcna- GFP, w; c855a-Gal4 (Bloomington Drosophila Stock Center, Indiana, USA), UAS-Histone2B::mRFP1 (from Y. Bellaiche). For clonal induction, we used hs- FLP, tub- Gal4, UAS-mCD8::GFP/CyO, FRT82B, tub- Gal80/TM6B and w; FRT82B (from B. Bello). For Notch misexpression experiment we used insc-Gal4; tub-Gal80ts (Bloomington) and UAS-Notch
(from S. Bray).
In vitro primary cell culture of larval brains
Primary cell cultures were obtained from wildtype and transgenic wandering third instar larvae. The cell culture protocol was adapted and modified after . The larvae were collected in PBS and their surface-sterilization achieved by immersing the larvae twice into 70% ethanol and three times into sterile water. Larval brains were dissected on sterile Petri dishes, in culture medium which is 90% L-Glutamine-supplemented Schneider`s medium (Invitrogen, Lucerne 6, Switzerland), 10% heat-inactivated Fetal Bovine Serum (HyClone Laboratories, Logan, UT, USA), 0.1% penicillin G (50–unit/ml)/streptomycin sulfate (50 μg/ml) (Gibco). The brain lobes and ventral nerve cord were collected in a saline similar to Rinaldini solution (800 mg NaCl, 20 mg KCl, 5 mg NaH2PO4H2O, 100 mg NaHCO3, 100 mg glucose, in 100 ml distilled water) and kept on ice, until the desired number of brains was collected. Dissected brains were washed three times in Rinaldini-like solution and then incubated in 0.5 mg sterile-filtered Collagenase solution type I (Sigma-Aldrich, Buchs, Switzerland)/ml Rinaldini-like solution, for 1 h, at room temperature. To remove the collagenase solution, three washes with culture medium were performed. A total of 10 μl of culture medium/brain were added and dissociation of brains into single cells was achieved by repeatedly flushing the brains through the pipette tip. For all of the protocol`s steps, we used siliconized Eppendorfs (Fisher Scientific, Wohlen, Switzerland) and pipette tips (VWR International, Dietikon, Switzerland). The cell suspension was plated into 96-well plates (40 μl cell suspension/well) (BD Falcon, Allschwil, Switzerland) and supplemented with culture medium (160 μl/well). Dissociated cells do not adhere and are free floating in culture medium. To achieve cell adhesion for cell differentiation experiments we cultured brain cell suspension on UV-treated 8-wells diagnostic Teflon slides (Thermo Scientific, Allschwil, Switzerland), coated with 15 μg sterile-filtered Concanavalin A/ml H2O (Sigma-Aldrich). A total of 40 μl cell suspension was placed in one well of the slide and supplemented with 40 μl culture medium. All cultures were kept in sterile wet boxes in an incubator (OKT Germany GmbH, Potsdam, Germany) at 25°C.
Immunofluorescent stainings and antibodies
For whole mount brain immunofluorescent labeling, larval brains were dissected in PBS (Gibco, Invitrogen). Brains were fixed for 30 min in 4% formaldehyde PBS solution (Sigma-Aldrich). Brains were washed in PBS-Triton 0.3% (Acros Organics, Basel, Switzerland) three times for 10 min. Non-specific binding sites in the tissue were blocked with 10% Normal Goat Serum (NGS) (Vector Laboratories, Servion, Switzerland) diluted in PBST for 30 min. Primary and secondary antibodies were diluted in PBST and 5% NGS. Primary antibodies were incubated overnight at 4°C and secondary antibodies at room temperature for 2 h. Brains were washed four times for 15 min. Brain samples were mounted in Vectashield mounting medium with DAPI (Vector Laboratories).
Primary brain culture immunofluorescent labeling was performed in a wet box, on 15 μg Concanavalin A/ml H2O-coated eight-well diagnostic Teflon slides (Thermo Scientific). A total of 40 μl cell suspension was placed in one well of the slide and allowed to settle down for 15 min. During this period cells start to adhere to the coated surface in order to perform immunostainings. The cells were fixed for 15 min in 4% formaldehyde and washed with PBST three times, every 2 min. 10% NGS treatment was applied for 30 min. Immunofluorescent labeling was performed as described for whole mount larval brains.
If not stated otherwise, antibody dilutions apply for immunofluorescent labelings of whole mount brains and cell cultures: rat and mouse anti-Elav (1:30, Developmental Studies Hybridoma Bank, DSHB, Iowa City, Iowa, USA), mouse anti-Pros (1:10, DSHB), mouse anti-Repo (1:10, DSHB), mouse anti-BrdU (1:200 for brains and 1:300 for primary brain culture staining, DSHB), mouse anti-FasII (1:30 on brains and 1:10 on cells, DSHB), sheep anti-GFP (1:1000, AbD, Serotec, Kidlington, UK), mouse anti-PH3 (1:1000, Cell Signaling Technology, Allschwil, Switzerland), rabbit anti phospho-Histone H3 (1:1000, Upstate Biotechnology, Lake Placid, NY, USA), rabbit anti Cleaved Caspase3 (1:200 for brains and 1:300 for primary brain culture staining, Cell Signaling Technology), rat anti-Deadpan (8:10, gift of C.Q. Doe), guinea pig anti-Deadpan (1:500, gift of J. Skeath), mouse anti-Discs large (1:100, DSHB), mouse anti-Neuroglian/BP104 (1:30, DSHB). Fluorescent labeled secondary antibodies raised in Donkey or Goat were used (Molecular Probes, Lucerne, Switzerland and Jackson ImmunoResearch, West Grove, PA, USA) at a dilution of 1:300 for whole brains and 1:400 for cultured cells.
BrdU labeling, clonal induction and Notch misexpression
Dissected L3 larval brains were incubated in 15 μg BrdU/ml of culture medium . For cell culture experiments, 15 μg of BrdU/ml culture medium was added. DNA was denatured in 2 N HCl for 30 min in whole brains and for 15 min in cultured cells. BrdU incorporation was detected by immunofluorescent labeling as described above.
To induce GFP labeled clones we used the MARCM (mosaic analysis with a repressible cell marker) system . Larvae with genotype hs- FLP; tubP- GAL4, UAS- mCD8::GFP/+; FRT82B, tub- GAL80/FRT82B were heat shocked for 1 h at 37°C and brains were dissociated at wandering third instar.
For Notch misexpression embryos of genotype w; insc-Gal4/+; tub-Gal80ts/UAS-Notch
developed at 18°C and freshly hatched larvae were transferred to 29°C to induce Notch gene expression in NBs. Notch misexpression cultures were prepared from wandering third instar larvae and incubated at 25°C.
Microscopy and image processing
A Leica TCS SP5 confocal microscope was used to collect Z stacks with optical sections at 0.5 to 1.5 μm intervals. Whole brains were imaged with a 20X objective and cultured cells were imaged with a 63X oil-immersion objective. To follow GFP tagged proteins in neurons we used DIC microscopy and the LAS AF software in our confocal. The Cell Counter plugin of ImageJ was used to analyze mitotic cells and apoptotic cells in single sections. Images were processed in ImageJ and Adobe Photoshop.