Rabbit anti ph3

Drosophila larvae were dissected in PBS, and larval brains were fixed in 3.7% formaldehyde with PBS + 0.3% Triton-100 (PBT) for 15 min. The samples were processed for immunostaining as previously described^{54 (link)}. Confocal images were taken from LSM710 and brightness and contrast were adjusted by Photoshop CS5.1. Primary antibodies used in this paper were guinea pig anti-Dpn (1:1000; J. Skeath), mouse anti-Mira (1:50, F. Matsuzaki), rabbit anti-Mira (1:500, W. Chia), mouse anti-Grh (1:10, AH Brand), rat anti-CD8 (1:250, Invitrogen, Cat#: MCD0800), rabbit anti-GFP (1:500, Molecular Probes, Cat#: A21311), rat anti-Elav (1:10, DSHB, Cat#: Rat-Elav-7E8A10), mouse anti-Pros (1:10, DSHB, Cat#: Prospero (MR1A)), mouse anti-Dac (1:5, DSHB, Cat#: mAbdac2-3), mouse anti-α-tubulin (1:200, Sigma, Cat#: T6199), guinea pig anti-Asl (1:200, G Rogers), mouse anti-γ-Tub (1:200, Sigma, Cat#: T5326), mouse Acetylated-Tub (1:100, Sigma, Cat#: T7451), rat anti-Kr (1:100, EADC, Cat#: 574), rabbit anti-Pdm1 (1:500, W. Chia & X. Yang), rabbit anti-PH3 (1:200, Sigma, Cat#: 06‐570), mouse anti-Dlg (1:200, DSHB, Cat#: 4F3 anti-discs large), rabbit anti-Klumpfuss (1:200, X. Yang) and guinea pig anti-Chro-C (1:500, this study). DNA was labelled by ToPro-3 (1:5000, Invitrogen, Cat#: T3605).

The following antibodies and dilutions were used: mouse anti-acetylated tubulin (Sigma; 1:500), rabbit anti-PH3 (Millipore; 1:500), mouse anti-SNAP25 (Synaptic Systems; 1:200), mouse anti-sodium channels (pan) clone K58/35 (Sigma; 1:500), mouse anti-SV2 (DSHB; 1:200), rabbit anti FIGQY (a gift from Matthew Rasband; 1:500). Primary antibodies were detected with appropriate secondary antibodies conjugated to either Alexa 488 or Alexa 568 (Molecular probes) at a 1:1000 dilution.
For immunostaining, embryos were fixed in 4% paraformaldehyde 1X PBS overnight at 4°C and stained as whole mounts. Sodium channels, SNAP25 and SV2 immunostainings were performed as described for Na_vCh staining in [33 (link)].
Images were taken on a Zeiss LSM510 system and a Leica SP8 confocal microscope.

For all immunostainings, zebrafish embryos were fixed in 4% PFA, washed in 1xPBT 3 times for 5 minutes and digested in a Trypsin- 2.5% EDTA solution for 30 minutes (on ice). Subsequently, they were washed in PBT 4 times for 10 minutes. The first solution change was performed on ice. To avoid unspecific binding of the antibodies, the embryos were blocked in a solution containing 10% goat serum, 1%BSA and 0.8% Triton in PBT for minimum 1 hour at room temperature. Blocking solution was replaced by the primary antibody mix chicken anti-GFP antibody [Life technologies, diluted 1:200)] and either rabbit anti-rx2 [Charles River (diluted 1:200)], rabbit anti-PH3 [Millipore, (diluted 1:400)] or mouse anti-PCNA antibody [Millipore (diluted 1:250)] and DAPI (diluted 1:1000) in the following solution: 1% goat serum, 1% BSA and 0.8% Triton. Embryos were incubated at 4°C for 2 overnights with primary antibodies. After washing in PBT for several hours, incubation with the secondary antibodies [anti- chicken antibody, Alexa 488 [Dianova (diluted 1:250)] and anti-rabbit antibody, Alexa 647 [Thermo Fischer (diluted1:250)] and DAPI, diluted 1:1000 in 1% goat serum, 1% BSA and 0.8% Triton solution was carried out for 2 overnights.

For standard immunostainings, intestines were dissected in 1 × PBS (10 mM NaH₂PO₄/Na₂HPO₄, 175 mM NaCl, pH 7.4), and fixed in 4% paraformaldehyde for 25 min at room temperature. Samples were washed with 1 x PBT (0.1% Triton X‐100 in 1 × PBS) and blocked in 3% BSA in 1 × PBT for 45 min. Primary antibodies were added to the samples and incubated at 4°C overnight. The following primary antibodies were used: mouse mAb anti‐Dl (C594.9B, 1:50, developed by S. Artavanis‐Tsakonas, DSHB), mouse mAb anti‐Prospero (MR1A, 1:100, developed by Chris Doe, DSHB), rabbit anti‐pH 3 (1:2000, Millipore), rabbit anti‐active caspase 3 (1:1000, Cell Signalling), and rabbit anti‐Yun (1:1000).²⁵ Primary antibodies were detected by fluorescent‐conjugated secondary antibodies from Jackson ImmunoResearch Laboratories. Secondary antibodies were incubated for 2 h at room temperature. DAPI (Sigma‐Aldrich; 0.1 μg/ml) was added after secondary antibody staining. The samples were mounted in mounting medium (70% glycerol containing 2.5% DABCO). All images were captured using a Zeiss inverted confocal microscope and were processed in Adobe Photoshop and Illustrator.

Primary antibodies used included rabbit anti-Kif2a (1: 1000, Abcam), rabbit anti-Tbr2 (1:500, Abcam), rabbit anti-Pax6 (1:200, Covance), mouse anti-beta III tubulin (TU20) (1:500, Abcam), mouse anti-nestin (1:500, Abcam), mouse anti-α tubulin (1:5000, CST), rat anti-Brdu (1:500, Abcam), rabbit and chicken anti-green fluorescent protein (1:500, GFP) (Invitrogen and Aves, respectively), rabbit anti-Ki67 (1:500, Thermo), rabbit anti-PH3 (1:300, Millipore), rabbit anti-cleaved caspase3 (1:300, Cell signaling), rabbit anti-AKT (1:500, CST), rabbit anti-p-AKT (1:500, CST), rabbit anti-β-catenin (1:200, Santa Cruz), mouse anti-GSK3β (1:200, Santa Cruz), and mouse anti-p-GSK3β (1:200, Santa Cruz). All the corresponding conjugated secondary antibodies were purchased from Invitrogen. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (Roche).