Rabbit cleaved caspase 3

The organoids were fixed and processed for immunostaining as is or after cryosectioning. Primary antibodies used for immunostaining were as follows: rabbit anti-SOX2 (Millipore), mouse anti-ISL1 (DSHB), rabbit anti-OLIG2 (IBL), mouse anti-NeuN (Chemicon), goat anti-NKX6.1 (R&D Systems), guinea pig anti-CHX10^{17 (link)}, rabbit anti-TUJ1 (BioLegend), mouse anti-TUJ1 (Millipore), rabbit cleaved-caspase3 (Cell Signaling Technology), goat anti-T (R&D Systems), rabbit anti-GFP (Abcam), goat anti-CHAT (Chemicon), and mouse anti-SMI-32 (BioLegend). After cell washes, samples were incubated with secondary antibodies and counterstained with Alexa Fluor® 488 phalloidin (Invitrogen) for axon labeling and Hoechst 33342 (Thermo Fisher Scientific) for nuclei staining.

Primary antibodies used in western blots were as follows: rabbit CASPASE-3 (#9662, Cell Signaling), rabbit cleaved CASPASE 3 (#9661, Cell Signaling), rabbit CDC6 (#3387, Cell Signaling), mouse GAPDH (G8140, US Biological, Swampscott, MA), rabbit MCM4 (BD Pharmingen, San Jose, CA), mouse PCNA (sc-56, Santa Cruiz Biotechnology), rabbit and mouse Yap (sc-15407 and sc-101199, Santa Cruz Biotechnology, Santa Cruz; #4912 Cell Signaling, Danvers, MA; #2060, Epitomics, Burlingame, CA). All antibodies were used at 1:1000 except GAPDH which was 1:2000.
Secondary antibodies were purchased from Jackson ImmunoResearch (West Grove, PA). HRP-conjugated Donkey anti-rabbit IgG (711–036–152) and HRP-conjugated Donkey anti-mouse IgG (715–035–150) were used at 1:4000 to 1:5000 for western blots.

Ovaries that were prepared for immunofluorescence were fixed in 4% methanol-free formaldehyde in PBS with 0.001% Triton-X for approximately 5 min. The samples were then washed in PBS with 0.1% Triton-X and blocked with 2% normal goat serum (NGS) for 2 hr. The primary antibody, rabbit cleaved caspase-3 (Cell Signaling (Beverly, Massachusetts) 5 A1E) at a concentration of 1:400, was incubated overnight at 4°C in 2% NGS. The secondary antibody used was Cy3 conjugated (Jackson Immunoresearch (West Grove, PA)) and used at a concentration of 1:150 during a 2-hr incubation at room temperature. This was followed by a 10-min nuclear stain by DAPI.
In order to assay whether feeding flies RU486 in The Fly Condo would be sufficient to turn on the MB gene switch construct, we placed flies into condos containing RU846⁺ food. Flies had the MB switch construct as well as a UAS-GFP nls construct, such that if the MB switch is activated, it should fluoresce with GFP. After a 24-hr period in The Fly Condo, adults were removed and fixed in 4% methanol-free formaldehyde in PBS with 0.001% Triton X overnight at 4°C. Brains were then dissected out of whole adults in PBS. The samples were then washed in PBS with 0.1% Triton X and stained with DNA staining with DAPI, for 10 min and mounted in Vectashield (Vector Laboratories (Burlingame, CA) Item No. H-1000) before imaging.

Rabbit anti-AAV VP1, VP2, VP3 (1:800, American Research Products, 03-61084), rabbit anti-NeuN (1:500, Cell Signaling, clone D3S3I), rabbit anti-GFAP (1:2000, Dako, Z0334), rabbit anti-TDP-43 (1:500, Proteintech, 18-280-1-AP), mouse anti-TDP43-phospho Ser409/410-1 (1:4000, Cosmo Bio, CAC-TIP-PTD-M01), mouse anti-Calbindin (1:1000, Sigma, C9848), mouse anti-p62 lck ligand (1:1000 for WB, 1:250 for ICC, BD Biosciences, clone 3, 610833), rabbit anti-α-tubulin (1:5000, Abcam, 4074), rabbit-cleaved-caspase 3 (1:1000, Cell Signaling, 9661), mouse anti-cleaved PARP (1:250, Cell Signaling, 9548), mouse anti-GAPDH (1:5000, Calbiochem, CB 1001), mouse anti-poly(GA) [1:500 for WB, 1:5000 for ICC, clone 5F2 (Mackenzie et al., 2013 (link)) kindly provided by Dieter Edbauer] were used at the dilutions listed.