Rabbit anti ph3

Third-instar larvae were dissected in phosphate-buffered saline (PBS). Wing imaginal discs were fixed in 4% paraformaldehyde at room temperature for 20 min and then washed three times for 5 min in PBS. Discs were blocked [2% BSA diluted in PBS 0.1% Triton X-100 (PBST)] for 45 min at room temperature, followed by incubation with primary antibodies (diluted in block) overnight at 4°C. Tissue was then washed three times in PBST and incubated with secondary antibodies (diluted in block) at room temperature for 1.5 h. Tissue was then washed a final three times in PBST and mounted in a 70% glycerol solution. The following primary antibodies and dilutions were used: mouse anti-β-galactosidase (1:2000, Promega, Madison, USA), mouse anti-Wg [1:100, Developmental Studies Hybridoma Bank (DSHB), Iowa City, USA], mouse anti-Cut (1:50 DSHB), mouse anti-N^ICD (1:50 DSHB), rat anti-Ci (1:50 DSHB), mouse anti-Delta (1:50 DSHB), mouse anti-Arm (1:50 DSHB), rabbit anti-cleaved Caspase 3 (1:100 Cell Signaling), rabbit anti-PH3 (1:200 Cell Signaling), guinea pig anti-Sens (1:500, a gift from Hugo Bellen, Dept. of Molecular and Human Genetics, Baylor College of Medicine, Houston, USA), mouse anti-Dll (1:300, a gift from Ian Duncan, Dept. of Biology, Washington University in St. Louis, St. Louis, USA). Secondary antibodies (Jackson ImmunoResearch, West Grove, USA) were used at a 1:200 dilution.

The primary antibodies included the following: rabbit anti-Ki67 (#15580, Abcam), mouse anti-beta III tubulin (TUJ1, #1637, Millipore), mouse anti-GFAP(#N206A/8, NeuroMab), mouse anti-NEUN (#MAB377, Millipore), chicken anti-MAP2 (#5392, Abcam), rabbit anti-PH3 (#9701, Cell Signaling), rabbit anti-TBR2 (#23345, Abcam), rabbit anti-cleaved caspase-3 (#9661, Cell Signaling), and rabbit anti-PAX6 (#PRB-278P, Covance). The secondary fluorochrome-conjugated antibodies were diluted 1:400 (donkey anti-mouse, donkey anti-rabbit, goat anti-mouse and goat anti-rabbit, goat anti-chicken (Invitrogen]). Nuclear counterstaining was performed with 4’, 6-diamidino-2-phenylindole dihydrochloride (DAPI) (Sigma-Aldrich, #B2261at 0.25 μg/μl). TUNEL staining was carried out using a kit purchased from Roche (#12156792910). Images were obtained using an LSM710 confocal microscope (Zeiss).

The following antibodies were used for cell division analyses; primary antibodies: rabbit anti-PH3 (Cell Signaling (Danvers, MA) 9701) 1:500; mouse anti-GFP (Cell Signaling 2955) 1:1000. Secondary antibodies: Alexa Fluor 594 donkey anti-rabbit ((A21207) Thermo Fisher Scientific, Waltham, MA) 1:1000; Alexa Fluor 488 donkey anti-mouse (A21202) 1:1000. Guts were dissected in ice cold PBS and immediately fixed in 4% formaldehyde for 15 min, serially dehydrated in MeOH, stored at -20°C, and subsequently stained. Guts were washed in 0.2% Triton-X / PBS, blocked in 5% bovine serum albumin / PBS, incubated in primary antibody overnight at 4°C and in secondary for 2 hr at RT. At least 10 guts per condition were mounted, scored and imaged as described above.

The following antibodies were used: mouse anti-Oct4 (Santa Cruz), rabbit anti-Nanog54 (link), anti-Sox2 (Calbiochem), rabbit anti-E-cad (extracellular)55 (link), mouse anti-E-cad (intracellular, BD Bioscience), mouse anti-N-cad (BD Bioscience), mouse anti-N-cad (Life Technologies), rat anti-ZO-156 (link), rat anti-pan-Laminin57 , goat anti-T-brachyury (Santa Cruz), goat anti-Sox17 (R&D Systems), rabbit anti-pH3 (Cell Signaling), rabbit anti-H3 (Abcam), mouse anti-β-catenin (BD Bioscience), mouse anti-Ezrin (Cell Signaling) and rabbit anti-pSMAD1-5-8 (Cell Signaling).

Drosophila S2 cells grown on cover slips, dissected ring glands and imaginal discs were processed as described [65] (link) and stained overnight at 4°C (tissues) or 2 h at room temperature (S2 cells) with the following antibodies: rat anti-Ecd^Nterm (1∶500, this study), guinea pig anti-Spok (1∶1000) and rabbit anti-Phm (1∶300) [33] (link), rabbit anti-cleaved Caspase-3 (1∶500, ASP175, Cell Signaling, #9661), rabbit anti-pH3 (1∶100, Cell Signaling, #9701), mouse anti-Flag M2 (1∶500, Sigma Aldrich), rabbit anti-c-Myc (1∶500, sc-789, Santa Cruz), and mouse anti-Lamin (1∶500, ADL67.10), mouse anti-EcR (1∶200, DDA2.7), and mouse anti-Fasciclin III (1∶300); the latter three from the Developmental Studies Hybridoma Bank (DSHB, Iowa). After washing, samples were incubated with corresponding secondary antibodies coupled to Cy3 or Cy5 (Jackson ImmunoResearch), counterstained with DAPI (0.5 µg/ml, Invitrogen) to visualize nuclei, and mounted in Dabco:Mowiol (Sigma-Aldrich).

Imaginal wing discs dissected from third instar larvae were collected in cold PBS and fixed in 4% paraformaldehyde. After proper washes, the wing discs were blocked in 10% horse serum, and stained with antibodies. The following antibodies were used: rabbit anti-pH3 (1:200 Cell Signaling Technology), rabbit anti-Cleaved Caspase-3 (1:200 Cell Signaling Technology) and mouse anti-Dlg (1:200). Secondary antibodies were anti-rabbit-Alexa (1:1000 Cell Signaling Technology) and anti-mouse-Cy3 (1:1000, Jackson Immuno Research).

The following antibodies and dilutions were used: rat anti-Twist (gift from A. Stathopoulos, California Institute of Technology, Pasadena, USA), mouse anti-β-Gal (1:1,000, Sigma), mouse anti-Hnt (1:200) and anti-Spi (1:500, Developmental Studies Hybridoma Bank); rabbit anti-PH3 (1:1000) and mouse anti-Cas3 (1:1000, Cell signaling) and TRITC- or CY5-conjugated secondary antibodies (1:500; Jackson Immunoresearch). Rabbit and mouse secondary antibodies Alexa 555 and rat Alexa 647 (1:500; Invitrogen) were used. 546-labeled Phalloidin was incubated with the tissue for 20 min to visualize cell membrane. Samples were mounted in medium (Vectashield) with DAPI to stain the nucleus and analyzed by confocal microscopy (Leica SP5). Fixation and staining followed standard protocols.