Ovaries were stained as described previously (Song et al., 2002 (link)). For nonpermeabilized staining, PBS was used instead of PBST (PBS + 0.1% Triton X-100). Extracellular Wg staining was performed according to a published protocol (Strigini and Cohen, 2000 (link)). In brief, dissected ovaries were incubated with anti-Wg (1:3) on ice for 40 min, rinsed thoroughly with cold PBS, and then fixed and costained according to conventional ovary staining procedures. Antibodies used were as follows: mouse anti-Dlp (against Dlp V523-Q702, clone 13G8, 1:5), mouse anti-Fas3 (7G10, 1:8), mouse anti-Hts (1B1, 1:5), mouse anti-lacZ (40-1a, 1:50), mouse anti-LamC (LC28.26, 1:20), mouse anti-Wg (4D4, 1:3), and rat anti-Vasa (1:10). These antibodies were from Developmental Studies Hybridoma Bank (DSHB). Other primary antibodies used were rabbit anti–phospho-Histone H3 (1:1,000; EMD Millipore), mouse anti-GFP (clone N86/38, used 1:5; UC Davis/National Institutes of Health NeuroMab Facility). Secondary antibodies used were Cy3- or FITC-conjugated goat anti–mouse IgG1 or IgG2a, Dylight 649–conjugated donkey anti–rat IgG (all from Jackson ImmunoResearch Laboratories, Inc.), goat anti–rabbit IgG, and goat anti–rat IgG conjugated to Alexa Fluor 488 (Molecular Probes). Stained samples were mounted in Vectashield (Vector Laboratories).
Rabbit anti phospho histone h3
Manufactured by Merck Group
Sourced in United States
Rabbit anti-phospho-Histone H3 is a primary antibody that recognizes the phosphorylated form of Histone H3 protein. Histone H3 phosphorylation is a post-translational modification associated with chromatin remodeling and cell division processes.
Automatically generated - may contain errors
Lab products found in correlation
Rabbit anti cleaved caspase 3, by Cell Signaling Technology (5 mentions)
Mouse anti brdu, by BD (4 mentions)
Mouse anti prospero, by Developmental Studies Hybridoma Bank (4 mentions)
Vectashield, by Vector Laboratories (3 mentions)
Rabbit anti gfp, by Thermo Fisher Scientific (3 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Fluorescent secondary antibody, by Jackson ImmunoResearch (2 mentions)
1 1 dioctadecyl 3 3 3 3 tetramethylindocarbocyanine, by Thermo Fisher Scientific (2 mentions)
5 bromo 2 deoxyuridine brdu, by Merck Group (2 mentions)
Rabbit anti gli3, by Santa Cruz Biotechnology (2 mentions)
Mouse anti tuj1, by Fortrea (2 mentions)
Rabbit anti cux1, by Santa Cruz Biotechnology (2 mentions)
Rabbit anti tbr2, by Abcam (2 mentions)
Rabbit anti pax6, by Fortrea (2 mentions)
Dharmafect 1 transfection reagent, by Horizon Discovery (2 mentions)
Rabbit anti gapdh, by Santa Cruz Biotechnology (2 mentions)
P38 mapk, by Cell Signaling Technology (2 mentions)
Phospho erk, by Cell Signaling Technology (2 mentions)
Alexa 488 labeled isolectin b4, by Thermo Fisher Scientific (2 mentions)
Phospho y951 vegfr2, by Cell Signaling Technology (2 mentions)
31 protocols using rabbit anti phospho histone h3
1
Drosophila Ovary Immunofluorescence Staining
2
Immunostaining of Drosophila Midguts
3
Immunostaining of Drosophila Midguts
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Female fly midguts were dissected in PBS and fixed for 1 h at room temperature in a solution containing: 4% formaldehyde in a pH 7.5 solution containing 100 mM glutamic acid, 25 mM KCl, 20 mM MgSO4, 4 mM sodium phosphate dibasic, 1 mM MgCl2. Guts were then washed briefly in PBS and blocked on a shaker with wash buffer (1 × PBS, 0.5% bovine serum albumin and 0.1% Triton X-100), first for 30 min at room temperature, followed by 1 h at 4°C. Rabbit anti-phospho-Histone H3 (EMD Millipore – 1:5000), mouse anti-Prospero (DSHB MR1A – 1:200), rabbit anti-PDM1 (gift from Xiaohang Yang at Zhejiang University – 1:300), or mouse anti-Delta (DSHB C594.9B – 1:100 dilution) primary antibodies were incubated overnight at 4C (1:5000,1:100 dilutions respectively). Fluorescent secondary antibodies from Jackson Immunoresearch were incubated for 4 h at room temperature (1:500 dilution). Antibodies were diluted in wash buffer. Hoechst was used to stain DNA. Fixed samples were imaged using a 3i W Spinning Disc Confocal.
4
Evaluating Karonudib's Impact on Cell Growth, Viability, and Apoptosis
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Measurement of relative cell growth (CellTiterGlo, 72 h, karonudib (0.0625–1 µM)), viability (propidium iodide, 72 h, karonudib (0.25–1 µM)) and apoptosis (Active Caspase-3, 24 h, karonudib (0.5 µM)) was performed as previously described20 (link). Proliferation (72 h, karonudib (0.25 µM) was performed using Cell Trace Violet (ThermoFisher Scientific). Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was performed together with cell cycle analysis after 6, 12 and 24 h with karonudib treatment (0.5 µM) as previously described20 (link),22 (link),23 (link). For cell cycle studies live/dead cell staining (near-IR dead cell stain kit L10119, Thermo Fisher Scientific) was performed prior to fixation. Antibodies: rabbit anti-phospho-histone H3 (pS10 #06-570 1:500; Merck), mouse anti-phospho-γ-histone H2AX (pS139 clone JWB301, #05-635 1:500; Merck), donkey anti-mouse IgG-Alexa488 (#715-545-150 1:500; Jackson Immunoresearch, West Grove, PA), and goat-anti-rabbit IgG-PE (1:500; Thermo). In addition we used biotin-16-dUTP (Merck), streptavidin-Cy5 (PA45001 1:400; GE Healthcare, UK) and Hoechst 33258 (2 µg/ml). Hoechst stained cells were stored at 4°C over night before analysis. Flow cytometry data were analyzed using the online Cytobank flow cytometry software (https://community.cytobank.org )24 or FlowJo v10.
5
Immunocytochemistry of Cellular Constructs
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Immunocytochemistry was performed on cellular constructs fixed in paraformaldehyde (Supplementary Methods). Primary antibodies were: 0.25% v/v rabbit anti-phospho-histone H3 (Merck Millipore: 06-570) or 0.67% v/v rabbit anti-SOX-9 (Merck Millipore: AB5535). Secondary donkey antibodies conjugated to Alexa Fluor 568 or 647 (Invitrogen: A10042 and A31573) were used with the SOX-9 and phospho-histone H3 antibodies, respectively. The immunostained constructs were imaged by confocal fluorescence microscopy (Zeiss LSM 710, Supplementary Methods).
6
Immunofluorescence Assay for Cell Markers
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The following reagents were used for immunofluorescence: rat anti-platelet endothelial cell adhesion molecule 1 (PECAM1; BD Pharmingen, 553370, 1:250-1000), mouse anti-α-smooth muscle actin-Cy3 (Sigma-Aldrich, C6198, 1:100-1000), rat anti-E-cadherin (Invitrogen, 53-3249-82, 1:500), rabbit anti-phosphohistone H3 (EMD Millipore, 06-570, 1:250) mouse anti-HOPX (E-1; Santa Cruz Biotechnology, sc-398703, 1:100), rabbit anti-SPC (Abcam, ab90716, 1:500). Prolong Gold mounting media (±DAPI) and Alexa Fluor fluorescent secondary antibodies (Alexa 488 donkey anti-rat A21208, Alexa 594 donkey anti-rabbit A21207, both diluted to 1:1000) were purchased from Invitrogen. The nuclear stain DRAQ5 (62251, 1:5000) was purchased from Thermo Fisher Scientific. Antibody titrations were performed on WT tissues to determine the optimal dilution for each tissue. Cells and explants were cultured in the following reagents: interferon-γ (R&D Systems, 485-MI-100), Dulbecco's modified Eagle's medium (DMEM; Corning Life Sciences, 10-013-CV), fetal bovine serum (FBS; Thermo Fisher Scientific), and penicillin-streptomycin (Thermo Fisher Scientific, 15140122).
7
Cortical Development Immunofluorescence Staining
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Perfusion, dissection, and immunofluorescence staining were conducted according to standard protocols as previously described [17 (link)]. The following are the antibodies used: mouse anti-BrdU (1:50 dilution; BD Pharmingen, Franklin Lakes, NJ, USA), rabbit anti-Cux1 (1:100 dilution; Santa Cruz Biotechnology, Dallas, TX, USA), rabbit anti-Phospho-Histone H3 (1:250 dilution; Millipore, Billerica, MA, USA), rabbit anti-Pax 6 (1:500 dilution; Covance, Princeton, NJ, USA), rabbit anti-Tbr2 (1:500 dilution; Abcam, Cambridge, UK), mouse anti-Tuj1 (1:500 dilution; Covance, Princeton, NJ, USA), rabbit anti-Gli3 (1:100 dilution; Santa Cruz Biotechnology, Dallas, TX, USA), and rabbit anti-Cleaved Caspase 3 (1:300 dilution; Cell Signaling, Madison, WI, USA).
For 5-bromo-2-deoxyuridine (BrdU, Sigma, St. Louis, MO, USA) labeling, pregnant dams were treated with 50 μg/g BrdU by intraperitoneal injection for 4 h prior to dissection at E16.5. DiI labeling was conducted by placing small crystals of the lipophilic tracer (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine; Invitrogen, Waltham, MA, USA) in the neocortex to target the upper layer (2/3) and then remained in 4% paraformaldehyde (PFA). After 6 weeks, brains were sectioned at 100 μm, counterstained with bisbenzimide, mounted, and imaged.
For 5-bromo-2-deoxyuridine (BrdU, Sigma, St. Louis, MO, USA) labeling, pregnant dams were treated with 50 μg/g BrdU by intraperitoneal injection for 4 h prior to dissection at E16.5. DiI labeling was conducted by placing small crystals of the lipophilic tracer (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine; Invitrogen, Waltham, MA, USA) in the neocortex to target the upper layer (2/3) and then remained in 4% paraformaldehyde (PFA). After 6 weeks, brains were sectioned at 100 μm, counterstained with bisbenzimide, mounted, and imaged.
8
Immunohistochemistry Protocol for CXCL13
9
Larval Immunostaining and Confocal Imaging
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Larvae were stained using methods modified from those previously described [29 (link)]. Briefly, larvae were fixed in 4% PFA in PBS overnight at 4°C. Larvae were washed in PBS + 0.25% Triton (PBT), incubated in 150mM Tris-HCl pH 9.0 for 15 min at 70°C, then washed in PBT. Larvae were permeabilized in 0.05% Trypsin-EDTA for 5 min on ice [30 ], washed in PBT, blocked in PBT containing 1% bovine serum albumin (BSA), 2% normal goat serum (NGS) and 1% dimethyl sulfoxide (DMSO), and then incubated in primary and secondary antibodies overnight at 4°C in PBT containing 1% BSA and 1% DMSO. Stained larvae were stored and mounted in Vectashield (Vector Laboratories) for imaging using confocal microscopy. Primary antibodies used were: mouse anti-GFP (JL-8, 1:200, BD Biosciences), rabbit anti-caspase-3 (1:500, BioRad), mouse anti-PCNA (pc10, 1:1000, Sigma), rabbit anti-phospho-histone H3 (1:500, Millipore). Secondary antibodies used were: goat anti-mouse Alexa 488 (1:500, Molecular Probes), goat anti-rabbit Alexa 594 (1:500, Molecular Probes), goat anti-mouse Alexa 594 (1:500, Molecular Probes).
10
Investigating EMCN Regulation of Angiogenesis
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Non-targeting control siRNA (siCtrl) and siRNA directed against EMCN (siEMCN; Dharmacon) were purchased as SMART pools. Dharmafect 1 transfection reagent (Dharmacon) was purchased for cell culture studies while in vivo studies used transit-TKO transfection reagent (Mirius Bio). A cDNA encoding full-length murine Emcn (provided by Dietmar Vestweber, Max-Planck-Institute, Germany) was cloned into a pShuttle vector and enhanced green fluorescent protein (EGFP) was cloned into pAdEasy. Adenoviral vectors were amplified in DH5alpha cells and purified plasmids were linearized and transfected into 293A cells, tittered by optical absorbance method, and expressed as plaque forming units (pfu) per ml. The EMCN adenovirus titer is 3.1 × 1010 pfu/mL and EGFP adenovirus titer is 1 × 1011 pfu/mL. The virus is distributed in the following formulation: 50 mM Tris-HCl, pH 7.4, 5 mM EDTA, 1.4 M CsCl, 50 mM NaCl, 0.5 mM MgCl2, and 25% glycerol. Retinas were stained with Alexa 488-labeled isolectin-B4 (Life Technologies), rat anti-EMCN (Santa Cruz Biotechnology), and rabbit anti-phospho-histone H3 (Millipore) while immunoblots were probed with antibodies against EMCN (Abcam), rabbit anti-GAPDH (Santa Cruz Biotechnology), phospho-Y951-VEGFR2, VEGFR2, phospho-p38-MAPK, p38-MAPK, phospho-ERK, ERK, phospho-FAK, and FAK (Cell Signaling).
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