Tissues were snap-frozen, embedded in OCT, and sectioned at 10 μm thickness. For histology, tissue sections were stained with hematoxylin and eosin. For immunostaining, tissue sections were post-fixed with 4% PFA for 10 mins and blocked with 2% BSA for 1 hour. The following primary antibodies were used: chicken anti-KRT14 (1:1000, gift from Dr. Xing Dai, UC Irvine), rabbit anti-LEF1(1:200, Cell Signaling), goat anti-PDGFRa (1:350, R&D), rat anti-CD31 (1:200, BD Bioscience), rat anti-CD45 (1:200, BD Bioscience), rabbit anti-Ki67 (1:1000, Cell Signaling), rabbit anti-PLIN (1:1000, Cell Signaling), rabbit anti-phospho-SMAD2 (1:250, Cell Signaling). DAPI was used for nuclei counterstaining. Tissue sections were visualized with an Olympus FV3000 confocal laser scanning microscope.
Rabbit anti lef1
Manufactured by Cell Signaling Technology
Sourced in United States
Rabbit anti-LEF1 is a primary antibody that recognizes the LEF1 (Lymphoid Enhancer Binding Factor 1) protein. LEF1 is a transcription factor that plays a role in the Wnt signaling pathway and is involved in the regulation of gene expression during embryonic development and cellular differentiation.
Automatically generated - may contain errors
Lab products found in correlation
Protein assay kit, by Bio-Rad (2 mentions)
Goat anti rabbit igg, by Jackson ImmunoResearch (2 mentions)
Enhanced chemiluminescence kit, by Thermo Fisher Scientific (2 mentions)
Mouse anti β actin, by Thermo Fisher Scientific (2 mentions)
Horseradish peroxidase hrp conjugated goat anti mouse igg, by Jackson ImmunoResearch (2 mentions)
Protein lysis buffer, by Thermo Fisher Scientific (2 mentions)
Rabbit anti β catenin, by Cell Signaling Technology (2 mentions)
Protease and phosphatase inhibitor, by Thermo Fisher Scientific (2 mentions)
Mouse anti np, by American Type Culture Collection (2 mentions)
Mouse anti ns1, by Santa Cruz Biotechnology (2 mentions)
Rabbit anti fzd4, by Cell Signaling Technology (2 mentions)
Mouse anti stat3, by Cell Signaling Technology (2 mentions)
Py705 stat3, by Cell Signaling Technology (2 mentions)
M o m kit, by Vector Laboratories (2 mentions)
Axioskop microscope, by Zeiss (2 mentions)
Immpact dab, by Vector Laboratories (2 mentions)
Goat anti lcn2, by R&D Systems (2 mentions)
Rabbit anti tcf4, by Cell Signaling Technology (2 mentions)
Rabbit anti tcf1, by Cell Signaling Technology (2 mentions)
Mouse anti β catenin, by Merck Group (2 mentions)
10 protocols using rabbit anti lef1
1
Multimodal Tissue Characterization
2
Protein Interactions in Developmental Signaling
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The following primary antibodies were used: mouse anti-β-catenin (Sigma-Aldrich Co., C-7207, 1:1,000); rabbit anti-β-catenin (Novus Biologicals, NBP1-32239, 1:200); goat anti-β-catenin (R&D Systems, AF-1329, 1:100); rabbit anti-KLF4 (Cell Signaling Technology, 4038S, 1:100); goat anti-KLF4 (R&D systems, AF-3158, 1:50); rabbit anti-LEF1 (Cell Signaling Technology, 2286, 1:100); rabbit anti-TCF3 (EMD Millipore, ABE414, 1:800; Cell Signaling Technology, 2883, 1:100); and anti-TCF4 (Cell Signaling Technology, 2565, 1:100). A Duolink PLA kit (Olink Bioscience, Uppsala, Sweden) was used according to the manufacturer's instructions with minor modifications. 4–6 μm frozen sections were fixed with 4% formaldehyde for 5 min, blocked in PBS/5% donkey serum/0.8% Triton X-100 for 2 h, incubated in primary antibodies overnight at 4 °C, washed in PBST, and incubated in PLA probe anti-goat plus or anti-mouse plus, and PLA probe anti-rabbit minus for 2 h. Ligation and amplification steps were performed following the manufacturer's protocol. The slides were washed twice in washing buffer and once in PBS for 20 min each. Slides were dried and mounted using Duolink mounting medium and photographed using a Leica Microsystems DM5500B fluorescent microscope. All assays were repeated with at least three biological replicates.
3
Western Blot Analysis of Viral Proteins
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The cells were lysed with an appropriate volume of protein lysis buffer (Thermo Fisher Scientific, Waltham, MA, USA) containing phosphatase and protease inhibitors (Thermo Fisher Scientific, Waltham, MA, USA). The protein concentration was then determined using the Bio‐Rad Protein Assay Kit (Bio‐Rad, CA, USA). Ten micrograms of each protein sample were electrophoresed by SDS‐PAGE and transferred onto a nitrocellulose membrane. Western blot analysis was performed using the following primary antibodies and dilutions: mouse anti‐NP (ATCC, HB‐65, 1:50), mouse anti‐NS1 (Santa Cruz biotechnology, Santa Cruz, CA, USA, 1:1000), mouse anti‐β‐actin (Thermo Fisher Scientific, 1:3000), rabbit anti‐β‐catenin (Cell Signaling, Beverly, MA, USA, 1:1000), rabbit anti‐LEF1 (Cell Signaling, 1:1000), and rabbit anti‐FZD4 (Cell Signaling, 1:1000). The membranes were incubated with primary antibodies overnight at 4°C, followed by incubation with horseradish peroxidase (HRP)‐conjugated goat anti‐mouse IgG (Jackson ImmunoResearch, PA, USA, 1:2000) or goat anti‐rabbit IgG (Jackson ImmunoResearch, 1:2000) at room temperature for 1 hr. Protein bands were visualised using the enhanced chemiluminescence kit (Thermo Fisher Scientific, Rockford, IL, USA).
4
Western Blot Analysis of Viral Proteins
5
Immunohistochemical and Immunofluorescence Analysis
6
Retinal Vascular Development Evaluation
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To evaluate the development of retinal vessels, retinae were dissected from the eyes and fixed in 4% PFA at 4 °C overnight, followed by a rinse in phosphate-buffered saline (PBS). After being blocked in blocking buffer containing 1% fetal bovine serum with 0.25% Triton X-100 for 1 h at room temperature, the retinae were incubated overnight with Isolectin B4 (I21413, Thermo Fisher Scientific, USA), and primary antibodies including rat anti-mouse Ter-119 (553670, BD Biosciences, USA), rat anti-mouse plasmalemma vesicle-associated protein (PLVAP)/MECA-32 (553849, BD Biosciences, USA), rabbit anti-LEF1 (76010, Cell Signaling Technologies, USA), rabbit anti-ERG (ab92513, Abcam, USA), and Alexa Fluor 488-conjugated Claudin-5 monoclonal antibody (352588, Thermo Fisher Scientific, USA). The retinae were washed 3 times in PBS followed by incubation with appropriate Alexa Fluor-conjugated secondary antibodies (A-11006, A32721, or A27018, Thermo Fisher Scientific, USA) for 4 h. The Click-iT EdU Alexa Fluor-488 Imaging Kit (C10337, Thermo Fisher Scientific, USA) was used in combination with ERG antibody to detect EdU positive endothelial cells in retinae of pups that were sacrificed 3 h after intraperitoneal EdU injection.
7
Immunohistochemical and Immunofluorescence Analysis
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For immunohistochemical detection, 5 μm paraffin sections were used; and endogenous biotin and peroxidase activity were blocked. The color was developed with DAB chromogen (ImmPACT DAB; VectorLabs, United States) and the sections were counterstained with hematoxylin.
Immunofluorescence analysis was performed as described22 (link). 6~8 μm frozen backskin sections were fixed in 4% PFA. For blocking, the MOM kit (Vector Laboratories, Burlingame, CA) was used for mouse monoclonal antibodies, and the following blocking buffer for all other antibodies: 10% normal donkey serum, 2% BSA, 2% fish skin gelatin, 2% Triton X-100 in PBS. Primary antibodies were used at the following concentration: guinea pig anti-Tcf3 (1:200, lab-generated), rabbit anti-Tcf4 (1:200; Cell Signalling), rabbit anti-Tcf1 (1:50; Cell Signaling), rabbit anti-Lef1 (1:150; Cell Signaling), mouse anti-myc tag (1:500; Zymed), mouse anti-β-catenin (Sigma), mouse anti-Stat3 (1:300; Cell Signaling), pY705-Stat3 (1:100; Cell Signaling), rat anti-cd104 (1:200; BD Biosciences) goat anti-Lcn2 (1:150;R&D Systems). Antigens were visualized with FITC-or RRX-conjugated secondary Abs (Jackson Labs). All images were acquired with Zeiss Axioskop microscope.
Immunofluorescence analysis was performed as described22 (link). 6~8 μm frozen backskin sections were fixed in 4% PFA. For blocking, the MOM kit (Vector Laboratories, Burlingame, CA) was used for mouse monoclonal antibodies, and the following blocking buffer for all other antibodies: 10% normal donkey serum, 2% BSA, 2% fish skin gelatin, 2% Triton X-100 in PBS. Primary antibodies were used at the following concentration: guinea pig anti-Tcf3 (1:200, lab-generated), rabbit anti-Tcf4 (1:200; Cell Signalling), rabbit anti-Tcf1 (1:50; Cell Signaling), rabbit anti-Lef1 (1:150; Cell Signaling), mouse anti-myc tag (1:500; Zymed), mouse anti-β-catenin (Sigma), mouse anti-Stat3 (1:300; Cell Signaling), pY705-Stat3 (1:100; Cell Signaling), rat anti-cd104 (1:200; BD Biosciences) goat anti-Lcn2 (1:150;R&D Systems). Antigens were visualized with FITC-or RRX-conjugated secondary Abs (Jackson Labs). All images were acquired with Zeiss Axioskop microscope.
8
Multimarker Immunofluorescence Characterization
9
Western Blot Analysis of Cranial Mesenchyme
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Enriched cranial mesenchyme was isolated as described above at E12.5 and protein was isolated as previously described [40 (link)]. Protein was isolated using RIPA buffer. Proteins were separated by SDS-PAGE using Mini-PROTEAN TGC gels (BioRad #456–1084, Hercules, CA, USA) or Mini-Protean TGX Stain-Free (BioRad #456-8084, Hercules, CA, USA). Western blots were performed with the following primary antibodies: rabbit-anti-p-ERK1/2 (1:500, Cell Signaling Technology 9101, Danvers, MA, USA), rabbit-anti-Erk1/2 (1:500, Cell Signaling Technology 9102, Danvers, MA, USA), and rabbit-anti-Lef1 (1:500, Cell Signaling Technology 2286, Danvers, MA, USA). Species-specific HRP-conjugated secondary antibodies were used at 1:10,000. Immunoblots were probed with anti-β-TUBULIN (1:400, Santa Cruz 9104, Dallas, TX, USA) as a loading control where indicated. Protein was detected using an Amersham ECL Western Blotting Analysis System (GE Healthcare RPN2109, Chicago, IL, USA), and developed onto film or imaged using a Biorad Chemidoc gel imager. Relative protein levels were quantified using Image J/Fiji to measure band intensity relative to the loading control (β-tubulin), or to total protein loaded per lane as measured by ImageLab analysis.
10
Cranial Mesenchyme Protein Isolation and Analysis
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Enriched cranial mesenchyme was isolated as described above at E12.5 and protein was isolated as previously described [40] . Protein was isolated using RIPA buffer. Proteins were separated by SDS-PAGE using Mini-PROTEAN TGC gels (BioRad #456-1084) or Mini-Protean TGX Stain-Free (BioRad #456-8084). Western Blots were performed with the following primary antibodies: rabbit-anti-p-ERK1/2 (1:500, Cell Signaling 9101), rabbit-anti-Erk1/2 (1:500, Cell Signaling 9102), rabbit-anti-Lef1 (1:500, Cell Signaling 2286). Species-specific HRP-conjugated secondary antibodies were used at 1:10,000. Immunoblots were probed with anti-β-TUBULIN (1:400, Santa Cruz 9104) as a loading control where indicated. Protein was detected using an Amersham ECL Western Blotting Analysis System (GE Healthcare RPN2109), and developed onto film or imaged using a Biorad Chemidoc gel imager. Relative protein levels were quantified using Image J/ Fiji to measure band intensity relative to the loading control (β-tubulin), or to total protein loaded per lane as measured by ImageLab analysis.
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