Immunofluorescence:Article Title: Normal cell cycle progression requires negative regulation of E2F1 by Groucho during S-phase and its relief at G2-phase.
Article Snippet: After three washes for 5 min with TBST, proteins were detected using Pierce ECL Western Blotting Substrate (ThermoFisher Scientific) in accordance with the manufacturer’s instructions. .. Primary antibodies used in this study were: rabbit anti-phospho-Histone 3 (1:100; Cell Signaling Technology, #9701); mouse anti-phospho-Histone 3 (1:100; Cell Signaling Technology, #9706); rabbit anti-pGro (1:100; Hasson et al., 2005; Cinnamon et al., 2008); mouse anti-Gro (diluted 1:1000 for immunofluorescence and 1:5000 for western blot analysis; generously contributed by Christos Delidakis, Institute of Molecular Biology and Biotechnology, Crete, Greece) (Delidakis et al., 1991); rat anti-total Gro (1:1000 for western blot analysis; Santa Cruz Biotechnology, sc-15786); rabbit anti-panTLE (1:100; Cell Signaling Technology, #4681); mouse anti-GFP [1:100; Developmental Studies Hybridoma Bank (DSHB), #8H11]; mouse anti-Cyclin A (1:20; DSHB, #A12); mouse anti-Cyclin B (1:20; DSHB, #F2F4); rabbit anti-Dcp1 (1:100; Cell Signaling Technology, #9578); rat anti-E2F1 (1:100; generously contributed by Stefan Thor, University of Queensland, Australia); and rabbit anti-HA (1:100; Cell Signaling Technology, #3724). .. Secondary antibodies were Alexa Fluor 488 AffiniPure Donkey Anti-Mouse IgG (1:400; Jackson ImmunoResearch 715-545-150), Rhodamine Red-X (RRX) AffiniPure Donkey Anti-Mouse IgG (1:400; Jackson ImmunoResearch 715-295-150), Cy5 AffiniPure Donkey Anti-Mouse IgG (1:400; Jackson ImmunoResearch 715-175-151) or Cy5 AffiniPure Goat Anti-Rabbit IgG (1:400; Jackson ImmunoResearch 111-175-144).
Article Title: Mosaic gastruloids reveal a temporal restriction for developmental cell competition.
Article Snippet: Alternatively, for high-throughput imaging, immunostained gastruloids were transferred in PBS−/− into 96-well PhenoPlates (Revvity/ PerkinElmer, 6055302) with up to 12 gastruloids per well, followed by automated Z-stack tile imaging on an Opera Phenix (Revvity/ PerkinElmer) in spinning disk configuration. .. The following antibodies were used for indirect immunofluorescence: mouse anti-p53 (1:4,000, clone 1C12; Cell Signaling, 2524S), rabbit anti-phospho-histone 3 (1:1,600, clone D2C8, Cell Signaling, 3377), rabbit anti-cleaved caspase 3 (1:5,000, clone 5A1E, Cell Signaling, 9664), rabbit anti-YAP (1:200, clone D8H1X, Cell Signaling, 14074), goat anti-E-Cadherin (1:1,000, R&D, AF648), rabbit anti-N-Cadherin (1:200, Abcam, ab18203), rabbit anti-FoxA2 (1:400, clone D56D6, Cell Signaling, 8186), goat anti-Tbx6 (1:200, R&D, AF4744), goat anti-Brachyury (1:100, R&D, AF2085), rabbit anti-Sox2 (1:200, Abcam, ab92494), goat anti-Otx2 (1:200, R&D, AF1979), rabbit anti-Sox3 (1:300, Invitrogen, PA5-35983), rat anti-Nanog (1:250, Invitrogen, 14-5761-80), goat anti-Pecam1 Alexa Fluor 488-conj. (1:400, R&D, FAB3628G-025), mouse anti-SSEA-1 Alexa Fluor 405-conj. (1:400, R&D, FAB2155V-100UG), donkey anti-Goat Alexa Fluor 488-conj. (1:500, Invitrogen, A-11055), goat anti-rabbit Alexa Fluor 488-conj. (1:500, Invitrogen, CA-11034), goat anti-rat Alexa Fluor 488-conj. (1:500, Invitrogen, A-11006) and donkey anti-mouse Alexa Fluor 488-conj. (1:500, Invitrogen, A-32766). ..
Article Title: Normal cell cycle progression requires negative regulation of E2F1 by Groucho during S phase and its relief at G2 phase
Article Snippet: After three washes for 5 min with TBST, proteins were detected using Pierce ECL Western Blotting Substrate (ThermoFisher Scientific) in accordance with the manufacturer's instructions. .. Primary antibodies used in this study were: rabbit anti-phospho-Histone 3 (1:100; Cell Signaling Technology, #9701); mouse anti-phospho-Histone 3 (1:100; Cell Signaling Technology, #9706); rabbit anti-pGro (1:100; ; ); mouse anti-Gro (diluted 1:1000 for immunofluorescence and 1:5000 for western blot analysis; generously contributed by Christos Delidakis, Institute of Molecular Biology and Biotechnology, Crete, Greece) ( ); rat anti-total Gro (1:1000 for western blot analysis; Santa Cruz Biotechnology, sc-15786); rabbit anti-panTLE (1:100; Cell Signaling Technology, #4681); mouse anti-GFP [1:100; Developmental Studies Hybridoma Bank (DSHB), #8H11]; mouse anti-Cyclin A (1:20; DSHB, #A12); mouse anti-Cyclin B (1:20; DSHB, #F2F4); rabbit anti-Dcp1 (1:100; Cell Signaling Technology, #9578); rat anti-E2F1 (1:100; generously contributed by Stefan Thor, University of Queensland, Australia); and rabbit anti-HA (1:100; Cell Signaling Technology, #3724). .. Secondary antibodies were Alexa Fluor 488 AffiniPure Donkey Anti-Mouse IgG (1:400; Jackson ImmunoResearch 715-545-150), Rhodamine Red-X (RRX) AffiniPure Donkey Anti-Mouse IgG (1:400; Jackson ImmunoResearch 715-295-150), Cy5 AffiniPure Donkey Anti-Mouse IgG (1:400; Jackson ImmunoResearch 715-175-151) or Cy5 AffiniPure Goat Anti-Rabbit IgG (1:400; Jackson ImmunoResearch 111-175-144).
Western Blot:Article Title: Normal cell cycle progression requires negative regulation of E2F1 by Groucho during S-phase and its relief at G2-phase.
Article Snippet: After three washes for 5 min with TBST, proteins were detected using Pierce ECL Western Blotting Substrate (ThermoFisher Scientific) in accordance with the manufacturer’s instructions. .. Primary antibodies used in this study were: rabbit anti-phospho-Histone 3 (1:100; Cell Signaling Technology, #9701); mouse anti-phospho-Histone 3 (1:100; Cell Signaling Technology, #9706); rabbit anti-pGro (1:100; Hasson et al., 2005; Cinnamon et al., 2008); mouse anti-Gro (diluted 1:1000 for immunofluorescence and 1:5000 for western blot analysis; generously contributed by Christos Delidakis, Institute of Molecular Biology and Biotechnology, Crete, Greece) (Delidakis et al., 1991); rat anti-total Gro (1:1000 for western blot analysis; Santa Cruz Biotechnology, sc-15786); rabbit anti-panTLE (1:100; Cell Signaling Technology, #4681); mouse anti-GFP [1:100; Developmental Studies Hybridoma Bank (DSHB), #8H11]; mouse anti-Cyclin A (1:20; DSHB, #A12); mouse anti-Cyclin B (1:20; DSHB, #F2F4); rabbit anti-Dcp1 (1:100; Cell Signaling Technology, #9578); rat anti-E2F1 (1:100; generously contributed by Stefan Thor, University of Queensland, Australia); and rabbit anti-HA (1:100; Cell Signaling Technology, #3724). .. Secondary antibodies were Alexa Fluor 488 AffiniPure Donkey Anti-Mouse IgG (1:400; Jackson ImmunoResearch 715-545-150), Rhodamine Red-X (RRX) AffiniPure Donkey Anti-Mouse IgG (1:400; Jackson ImmunoResearch 715-295-150), Cy5 AffiniPure Donkey Anti-Mouse IgG (1:400; Jackson ImmunoResearch 715-175-151) or Cy5 AffiniPure Goat Anti-Rabbit IgG (1:400; Jackson ImmunoResearch 111-175-144).
Article Title: Normal cell cycle progression requires negative regulation of E2F1 by Groucho during S phase and its relief at G2 phase
Article Snippet: After three washes for 5 min with TBST, proteins were detected using Pierce ECL Western Blotting Substrate (ThermoFisher Scientific) in accordance with the manufacturer's instructions. .. Primary antibodies used in this study were: rabbit anti-phospho-Histone 3 (1:100; Cell Signaling Technology, #9701); mouse anti-phospho-Histone 3 (1:100; Cell Signaling Technology, #9706); rabbit anti-pGro (1:100; ; ); mouse anti-Gro (diluted 1:1000 for immunofluorescence and 1:5000 for western blot analysis; generously contributed by Christos Delidakis, Institute of Molecular Biology and Biotechnology, Crete, Greece) ( ); rat anti-total Gro (1:1000 for western blot analysis; Santa Cruz Biotechnology, sc-15786); rabbit anti-panTLE (1:100; Cell Signaling Technology, #4681); mouse anti-GFP [1:100; Developmental Studies Hybridoma Bank (DSHB), #8H11]; mouse anti-Cyclin A (1:20; DSHB, #A12); mouse anti-Cyclin B (1:20; DSHB, #F2F4); rabbit anti-Dcp1 (1:100; Cell Signaling Technology, #9578); rat anti-E2F1 (1:100; generously contributed by Stefan Thor, University of Queensland, Australia); and rabbit anti-HA (1:100; Cell Signaling Technology, #3724). .. Secondary antibodies were Alexa Fluor 488 AffiniPure Donkey Anti-Mouse IgG (1:400; Jackson ImmunoResearch 715-545-150), Rhodamine Red-X (RRX) AffiniPure Donkey Anti-Mouse IgG (1:400; Jackson ImmunoResearch 715-295-150), Cy5 AffiniPure Donkey Anti-Mouse IgG (1:400; Jackson ImmunoResearch 715-175-151) or Cy5 AffiniPure Goat Anti-Rabbit IgG (1:400; Jackson ImmunoResearch 111-175-144).
Flow Cytometry:Article Title: Mosaic gastruloids reveal a temporal restriction for developmental cell competition.
Article Snippet: After three more washes with staining buffer, cells were analysed using a BD Bioscience LSRFortessa system. .. Antibodies used for flow cytometry: mouse anti-p53 (1:2,000, clone 1C12, Cell Signaling, 2524S), rabbit anti-phospho-histone 3 (1:1,600, clone D2C8, Cell Signaling, 3377) and rabbit anti-cleaved caspase 3 (1:5,000, clone 5A1E, Cell Signaling, 9664). .. Apoptosis and necrosis staining was performed using a commercial Annexin V kit (Invitrogen, A13201) in combination with DAPI staining.
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