Incubation:Article Title: Brain vascular stability relies on PAK2–cilia–PDGF-BB–HSPGs on basolateral side of endothelium
Article Snippet: .. After incubation, EC tubes were washed with PBS for 20 min, followed by perfusion of the primary antibody solution containing ARL13B (Cat# 17711-1-AP; Proteintech) and acetylated α-tubulin (Cat# 32-2700; Thermo Fisher Scientific) both at a dilution of 1:200 (20 min), followed by a 48-h incubation at 4°C. .. EC tubes were then washed with PBS for 20 min, and the previous step was repeated with secondary antibody (Cat# ab150088; ab150077; Abcam) solution (1:300) containing DAPI (1:300) for nuclear staining.
Article Title: Loss of SPECC1L in cranial neural crest cells results in increased hedgehog signaling and frontonasal dysplasia
Article Snippet: Sodium citrate buffer consisted of sodium citrate dihydrate (Sigma, #S4641), pH 6.0, and 0.05% Tween-20 (Fisher, #BP337). .. Blocking with 10% goat serum in PBS (Omega Scientific, DG-11) was performed at RT for 1 h. Sections were then incubated overnight at 4 °C with primary antibodies: SPECC1L N-terminus (1:250, Proteintech, 25390-1-AP), ARL13B (1:300, Proteintech, 17711-1-AP), Ki-67 (1:500, Cell Signaling, 12202), GLI1 (1:100, Cell Signaling, 2553S), GLI3 (1:100, R&D System, AF3690), Non-phospho (Active) β-catenin (1:500, Cell Signaling, 8814S), SOX10 (1:50, Proteintech, 10422-1-AP), SOX10 (1:30, Santa Cruz, sc-365692), β-catenin (1:250, Proteintech, 2677S), or E-cadherin (1:400, Cell Signaling, 14472S). .. Secondary antibodies and stains were incubated for an hour at RT: Goat anti-rabbit IgG (H + L) Alexa 647 (1:500, Invitrogen, A21245), Donkey anti-goat IgG (H + L) Alexa 647 (1:500, Invitrogen, A21447), Goat anti-rabbit IgG (H + L) Alexa 488 (1:500, Invitrogen, A11008), Goat anti-mouse IgG1 Alexa 488 (1:500, Invitrogen, A21121) or Acti-stain 670 phalloidin (1:200, Cytoskeleton, PHDN1-A).
Article Title: Brain vascular stability relies on PAK2–cilia–PDGF-BB–HSPGs on basolateral side of endothelium
Article Snippet: Fixed cells were washed again with 1X PBS before permeabilization with 0.1% Triton X-100 (Cat# 1610407; Bio-Rad) for 15 min. .. This was followed by blocking in 4% BSA in PBS and overnight incubation with primary antibodies of ARL13B (Cat# 17711-1-AP, dilution 1:500; Proteintech) and PDGF-BB (Cat# NBP1-58279, dilution 1:500; Novus Biologicals). .. Cells were again washed with 1X PBS and incubated with Alexa Fluor 488 anti-rabbit (Cat#A21206; Invitrogen) (1:500) and Alexa Fluor 568 anti-rabbit (Cat# A10042; Invitrogen), for 90 min at RT, and washed before mounting with DAPI (Cat# LS-J1033-10; LifeSpan Biosciences) and imaged using a Zeiss confocal microscope at a magnification of 63X.
Staining:Article Title: Bi-allelic INTU variants define a ciliopathy disorder characterized by orofacial, digital, and cardiac anomalies
Article Snippet: .. After 48 hours, cells were stained for ARL13b (1:500, Proteintech 17711-1-AP, Rosemont, IL) and γ-Tubulin (1:500, Millipore-Sigma T6557, Burlington, MA) after blocking with 4% normal goat serum. .. Secondary antibodies used were Alexa Fluor 488 Gt α-Rb (1:1500, Invitrogen A11001, Waltham, MA) and Alexa Fluor 555 Gt α-Rb (1:1500, Invitrogen A21428, Waltham, MA) before counterstaining with 5μg/mL DAPI (Sigma-Aldrich D9542, Burlington, MA) and mounted in Prolong Gold Medium (Invitrogen P36930, Waltham, MA).
Article Title: Bi-allelic INTU variants define a ciliopathy disorder characterized by orofacial, digital, and cardiac anomalies
Article Snippet: .. After 48 hours, cells were stained for ARL13b (1:500, Proteintech 17711-1-AP, Rosemont, IL) and γ-Tubulin (1:500, Millipore-Sigma T6557, Burlington, MA) after blocking with 4% normal goat serum. .. Secondary antibodies used were Alexa Fluor 488 Gt α-Rb (1:1500, Invitrogen A11001, Waltham, MA) and Alexa Fluor 555 Gt α-Rb (1:1500, Invitrogen A21428, Waltham, MA) before counterstaining with 5μg/mL DAPI (Sigma-Aldrich D9542, Burlington, MA) and mounted in Prolong Gold Medium (Invitrogen P36930, Waltham, MA).
Blocking Assay:Article Title: Bi-allelic INTU variants define a ciliopathy disorder characterized by orofacial, digital, and cardiac anomalies
Article Snippet: .. After 48 hours, cells were stained for ARL13b (1:500, Proteintech 17711-1-AP, Rosemont, IL) and γ-Tubulin (1:500, Millipore-Sigma T6557, Burlington, MA) after blocking with 4% normal goat serum. .. Secondary antibodies used were Alexa Fluor 488 Gt α-Rb (1:1500, Invitrogen A11001, Waltham, MA) and Alexa Fluor 555 Gt α-Rb (1:1500, Invitrogen A21428, Waltham, MA) before counterstaining with 5μg/mL DAPI (Sigma-Aldrich D9542, Burlington, MA) and mounted in Prolong Gold Medium (Invitrogen P36930, Waltham, MA).
Article Title: Loss of SPECC1L in cranial neural crest cells results in increased hedgehog signaling and frontonasal dysplasia
Article Snippet: Sodium citrate buffer consisted of sodium citrate dihydrate (Sigma, #S4641), pH 6.0, and 0.05% Tween-20 (Fisher, #BP337). .. Blocking with 10% goat serum in PBS (Omega Scientific, DG-11) was performed at RT for 1 h. Sections were then incubated overnight at 4 °C with primary antibodies: SPECC1L N-terminus (1:250, Proteintech, 25390-1-AP), ARL13B (1:300, Proteintech, 17711-1-AP), Ki-67 (1:500, Cell Signaling, 12202), GLI1 (1:100, Cell Signaling, 2553S), GLI3 (1:100, R&D System, AF3690), Non-phospho (Active) β-catenin (1:500, Cell Signaling, 8814S), SOX10 (1:50, Proteintech, 10422-1-AP), SOX10 (1:30, Santa Cruz, sc-365692), β-catenin (1:250, Proteintech, 2677S), or E-cadherin (1:400, Cell Signaling, 14472S). .. Secondary antibodies and stains were incubated for an hour at RT: Goat anti-rabbit IgG (H + L) Alexa 647 (1:500, Invitrogen, A21245), Donkey anti-goat IgG (H + L) Alexa 647 (1:500, Invitrogen, A21447), Goat anti-rabbit IgG (H + L) Alexa 488 (1:500, Invitrogen, A11008), Goat anti-mouse IgG1 Alexa 488 (1:500, Invitrogen, A21121) or Acti-stain 670 phalloidin (1:200, Cytoskeleton, PHDN1-A).
Article Title: Bi-allelic INTU variants define a ciliopathy disorder characterized by orofacial, digital, and cardiac anomalies
Article Snippet: .. After 48 hours, cells were stained for ARL13b (1:500, Proteintech 17711-1-AP, Rosemont, IL) and γ-Tubulin (1:500, Millipore-Sigma T6557, Burlington, MA) after blocking with 4% normal goat serum. .. Secondary antibodies used were Alexa Fluor 488 Gt α-Rb (1:1500, Invitrogen A11001, Waltham, MA) and Alexa Fluor 555 Gt α-Rb (1:1500, Invitrogen A21428, Waltham, MA) before counterstaining with 5μg/mL DAPI (Sigma-Aldrich D9542, Burlington, MA) and mounted in Prolong Gold Medium (Invitrogen P36930, Waltham, MA).
Article Title: Brain vascular stability relies on PAK2–cilia–PDGF-BB–HSPGs on basolateral side of endothelium
Article Snippet: Fixed cells were washed again with 1X PBS before permeabilization with 0.1% Triton X-100 (Cat# 1610407; Bio-Rad) for 15 min. .. This was followed by blocking in 4% BSA in PBS and overnight incubation with primary antibodies of ARL13B (Cat# 17711-1-AP, dilution 1:500; Proteintech) and PDGF-BB (Cat# NBP1-58279, dilution 1:500; Novus Biologicals). .. Cells were again washed with 1X PBS and incubated with Alexa Fluor 488 anti-rabbit (Cat#A21206; Invitrogen) (1:500) and Alexa Fluor 568 anti-rabbit (Cat# A10042; Invitrogen), for 90 min at RT, and washed before mounting with DAPI (Cat# LS-J1033-10; LifeSpan Biosciences) and imaged using a Zeiss confocal microscope at a magnification of 63X.
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