Two‐photon imaging with z‐compensation was performed to detect the fluorophore staining. Blood vessel endothelial cells were labelled with Alexa Fluor 594‐conjugated rat anti‐mouse CD31 (1 μg/ml, clone MEC13:3; Biolegend) (excited @ 760 nm) or Alexa Fluor 647‐conjugated rat anti‐mouse CD144 (2.5 μg/ml, clone BV13, Biolegend), and macrophages were labelled with Alexa Fluor 488‐conjugated anti‐mouse CD68 (2.5 μg/ml, clone FA‐11; Biolegend—excited @ 710 nm). Multiphoton images were generated on a Leica SP8 MP/CLSM system (Leica Microsystems).
Mec13
Manufactured by BioLegend
Sourced in United States
The MEC13.3 is a lab equipment product from BioLegend. It serves as a core function to facilitate research activities in controlled laboratory environments.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Secondary rabbit envision, by Agilent Technologies (2 mentions)
Horse serum, by Biocare Medical (2 mentions)
Decloaker, by Biocare Medical (2 mentions)
Rabbit anti hif 1α, by Abcam (2 mentions)
Citrate buffer ph 6, by Biocare Medical (2 mentions)
67 antibody d2h10, by Cell Signaling Technology (2 mentions)
Mec14, by Novus Biologicals (2 mentions)
Rat on mouse hrp polymer and probe, by Biocare Medical (2 mentions)
Lsm 780, by Zeiss (2 mentions)
Attune nxt, by Thermo Fisher Scientific (2 mentions)
Leica sp8 mp clsm system, by Leica Microsystems (1 mentions)
Cm1950, by Leica camera (1 mentions)
Yts156, by BioLegend (1 mentions)
Zen microscope software, by Zeiss (1 mentions)
Easysep, by STEMCELL (1 mentions)
Macs column, by Miltenyi Biotec (1 mentions)
Anti cd31 pe, by BD (1 mentions)
Facsaria fusion cell sorter, by BD (1 mentions)
Dispase 1, by Roche (1 mentions)
20 protocols using mec13
1
Multicolor Vascular Imaging Protocol
2
Intravital Imaging of Bacterial Capture
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Intravital imaging of mice was performed by confocal microscopy. For imaging of bacterial capture, hepatic macrophages and endothelial cells were visualized by intravenous injection of anti–F4/80-BV421 (5 μg/mouse, BM8, BioLegend) and anti–CD31-phycoerythrin (anti–CD31-PE) (5 μg/mouse, MEC13.3, BioLegend) antibodies. Mice were anesthetized by intraperitoneal injection of ketamine/medetomidine (125/1 mg/kg). After a midabdominal incision was made, the mouse was placed in a right lateral position, and the largest liver lobe was externalized on a coverslip attached to the microscope stage. The liver was covered with saline-moistened lab wipes to restrict movement, and the microscope heating chamber was kept at 37°C to maintain mouse body temperature. The liver was then imaged with an inverted Leica TCS SP5 confocal microscope with the Leica DM6000 Confocal Fixed Stage system. Bacteria that remained associated with KCs for more than 2 minutes were considered captured. Time-lapse images were recorded for up to 20 minutes after infection. A total of 5–6 field of view (FOV) images were recorded for each mouse under a ×20 microscope objective, and their average was considered 1 FOV. Videos and images were processed and analyzed with Imaris 8 software (Bitplane).
3
Immunofluorescent Analysis of Mouse Muscle
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Mouse gastrocnemius muscles were harvested, embedded in O.C.T. (optimal cutting temperature) compound, and immediately frozen on dry ice. Tissues were cut into 5-μm sections in a cryomicrotome (Leica CM1950). Sections were blocked with 5% BSA and then stained with anti–CD8β-PE (BioLegend, clone YTS156.7.7, catalog no. 126608, 1:100 dilution). Sections were then fixed and permeabilized with eBioscience Foxp3/transcription factor staining buffer set (catalog no. 00-5523) and stained with anti-dystrophin [Abcam, polyclonal rabbit immunoglobulin G (IgG), catalog no. ab15277, 1:50 dilution] and anti-mouse CD31-FITC (BioLegend, clone MEC13.3, catalog no. 102506, 1:50 dilution). After washing with PBST, the sections were stained with a secondary antibody (BioLegend, anti-rabbit IgG AF647, catalog no. 406414, 1:200 dilution) and DAPI (4′,6-diamidino-2-phenylindole). We imaged the sections using a Zeiss LSM710 confocal microscope. The fluorescent images were processed with the Zeiss ZEN microscope software.
4
Isolation of Fibroblastic Reticular Cells
5
Gastruloid Dissociation and Flow Cytometry
6
Conditional Ela Knockout Mouse Generation
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Example 54
Construction of Conditional Ela Knockout Mouse
A linearized targeting vector containing homology arms and exon 2 of ELABELA, flanked by lox-p sites together with a neo cassette flanked by FRT sites was electroporated into 129Sv mouse embryonic stems cells. Targeting was verified by southern blotting. Verified clones were transfected with Flp recombinase to remove the neo cassette, and were subsequently injected into C57BL6 recipient blastocysts and implanted into pseudopregnant females. F0 founders with >50% chimerism were backcrossed to C57BL6. The F1 generation, upon confirmation of stable ELAFlox germline transmission, were crossed with ZP3-cre recombinase-containing transgenic mice to remove Exon 2 to generate the ElaΔ null allele. ElaΔ/+ heterozygous mice were subsequently backcrossed for 8 generations to C57BL6 before phenotypic analysis.
Phenotypic Characterization of Ela-Deficient Embryos
ElaΔ/+ mice were intercrossed to generate ElaΔ/Δ, ElaΔ/+ and wildtype embryos for phenotypic analysis. Embryos were harvested at e10.5. Yolk sacs and embryos were fixed for 1 hour with 4% PFA, followed by immunohistochemistry for Pecam-1 (MEC13.3, Biolegend). Embryos and yolk sacs were also paraffin-embedded and sectioned for hematoxylin and eosin (H&E) staining to observe the morphology of cardiovascular structures.
7
Immunohistochemical Analysis of Tumor Samples
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Tumor sections were preserved in OCT compound, frozen and sectioned. 5 μm thick sections were fixed in 35/65 methanol/acetone. CD31 was detected with anti-mouse CD31 antibody (MEC13.3, Biolegend, San Diego CA). For the remaining IHC staining, Formalin-fixed tissue samples were paraffinized and sectioned by University of Iowa Comparative Pathology Core. Sections were then de-paraffinized in a series of xylene washes and rehydrated with ethanol and water. Antigen retrieval was accomplished by immersing slides in Citrate Buffer pH 6.0 in a decloaker (Biocare Medical, Concord CA). Tissue samples were incubated with 3% hydrogen peroxide followed by blocking with horse serum (Biocare Medical). Slides were then incubated with rabbit anti-mouse Ki-67 antibody (D2H10, Cell Signaling Technology), rat anti-CD34 antibody (MEC14.7, Novus Biologicals, Littleton CO) or rabbit anti-HIF1α (polyclonal, Abcam, Cambridge UK). Slides were then stained with Rat-on-mouse HRP polymer and probe (Biocare,) or secondary rabbit envision (DAKO, Santa Clara CA). The slides were developed with 3,3’-diaminobenzadine (DAB and 0.3% H2O2 in PBS) developing buffer, followed by counterstaining with hematoxylin. Positive staining was quantified using Image J software. All analysis of IHC was done blinded in regards to group allocation.
8
Immunofluorescent Labeling of Liver Cells
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For immunofluorescence labeling, liver sections were (co-)stained with antibodies directed against GS (1:1000; ab73593, Abcam), SOX9 (1/100; AB5535, Millipore), F4/80 (1:100; MCA497RT, BioRad), HNF4ɑ (1/100; sc6556, Santa Cruz) or CD31 (1:200; MEC13.3, BioLegend). Secondary antibodies were either anti-rabbit AlexaFluor 488; or anti-rabbit, anti-goat or anti-rat AlexaFluor 647 (1:1000, Invitrogen). Hoechst (1:10 000, 62249, Thermo Scientific) was used to counterstain the nuclei. Liver sections were imaged using the Zeiss AxioImager.Z2 microscope.
9
Conditional Ela Knockout Mouse Generation
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Example 54
Construction of Conditional Ela Knockout Mouse
A linearized targeting vector containing homology arms and exon 2 of Elabela, flanked by lox-p sites together with a neo cassette flanked by FRT sites was electroporated into 129Sv mouse embryonic stems cells. Targeting was verified by southern blotting. Verified clones were transfected with Flp recombinase to remove the neo cassette, and were subsequently injected into C57BL6 recipient blastocysts and implanted into pseudopregnant females. F0 founders with >50% chimerism were backcrossed to C57BL6. The F1 generation, upon confirmation of stable ELAFlax germline transmission, were crossed with ZP3-cre recombinase-containing transgenic mice to remove Exon 2 to generate the Ela4 null allele. ElaΔ/+ heterozygous mice were subsequently backcrossed for 8 generations to C57BL6 before phenotypic analysis.
Phenotypic Characterization of Ela-Deficient Embryos
ElaΔ/+ mice were intercrossed to generate ElaΔ/Δ, ElaΔ/+ and wildtype embryos for phenotypic analysis. Embryos were harvested at e10.5. Yolk sacs and embryos were fixed for 1 hour with 4% PFA, followed by immunohistochemistry for Pecam-1 (MEC13.3, Biolegend). Embryos and yolk sacs were also paraffin-embedded and sectioned for hematoxylin and eosin (H&E) staining to observe the morphology of cardiovascular structures.
10
Endothelial Cell Isolation via FACS
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Embryos were incubated with 0.3% collagenase (Cat # 038-22361; Wako, Osaka, Japan) at 37 °C for 15 min. After washing, single cells were exposed to allophycocyanin-conjugated anti-mouse Pecam1 antibody (MEC13.3, Cat #102510; BioLegend, San Diego, CA, USA). Cells were analyzed by FACS calibur (Becton Dickinson, San Jose, CA, USA) and the data were processed by the FlowJo software (TreeStar, Ashland, OR, USA).
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