Mice were perfused with 10 ml PBS under terminal anaesthesia, tissue samples were minced into small pieces and incubated in digestion buffer containing 1× PBS, collagenase D (1 mg ml−1, Sigma-Aldrich), dispase (2.4 mg ml−1, Thermo Fisher Scientific), DNase (0.2 mg ml−1, Sigma-Aldrich) and 3% heat-inactivated fetal bovine serum (FBS, Invitrogen) for 30 min at 37 °C. Cells suspensions were dissociated and passed through a 100 μm cell strainer (BD) and resuspended in 50 μl of blocking buffer containing 1× PBS, 0.5% BSA, 2 mM EDTA, anti-mouse CD16/32 (1:100), 5% normal rat, 5% normal mouse and 5% normal rabbit serum (Jackson ImmunoResearch) for 15 min at 4 °C. The samples were stained with the indicated antibodies (a list of which is provided in Supplementary Data 5 ; 1:200) for 30 min at 4 °C. Flow cytometry was performed using the BD Biosciences LSR Fortessa flow cytometer with Diva software. All data were analysed using FlowJo v.10.6 (Tree Star).
Normal rabbit serum
Manufactured by Jackson ImmunoResearch
Sourced in United States
Normal rabbit serum is a biological reagent derived from healthy rabbits. It contains a natural mixture of proteins, antibodies, and other components present in the rabbit's circulatory system. This product can be used as a control or baseline reference in various immunological and biochemical applications.
Automatically generated - may contain errors
Lab products found in correlation
Paraformaldehyde (pfa), by Thermo Fisher Scientific (3 mentions)
Dispase, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Cellsens software, by Olympus (2 mentions)
Ix3 confocal microscope, by Olympus (2 mentions)
Rabbit anti dog igg h l fitc antibody, by Jackson ImmunoResearch (2 mentions)
Lsrfortessa flow cytometer, by BD (1 mentions)
Dnase, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Collagenase d, by Merck Group (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Operetta cls high content imaging system, by PerkinElmer (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Mowiol 4 88, by Merck Group (1 mentions)
Donkey serum, by Jackson ImmunoResearch (1 mentions)
Sc 10368, by Santa Cruz Biotechnology (1 mentions)
Sc 1488, by Santa Cruz Biotechnology (1 mentions)
Alexa fluor 647 labeling kit, by Thermo Fisher Scientific (1 mentions)
Vectastain elite abc kit, by Vector Laboratories (1 mentions)
Aec substrate kit, by Vector Laboratories (1 mentions)
9 protocols using normal rabbit serum
1
Tissue Digestion and Cell Isolation
2
Whole-Mount Immunofluorescence Staining of Enteroids
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Enteroids were fixed with 4% paraformaldehyde at the end of differentiation and recovered from the matrix for whole-mount immunofluorescence staining.52 Antigen-retrieval was performed using 10 mM sodium citrate pH 6.0 for 30 min at 80°C followed by pre-incubation (2 h) with 10% donkey serum (Jackson ImmunoResearch) and 0.3% Triton X-100 (Sigma-Aldrich). Enteroids were then incubated overnight at 4°C with goat anti-chromogranin A (1:250; sc-1488, Santa Cruz), goat anti-ghrelin (1:250; sc-10368, Santa Cruz), rabbit anti-GLP-1 (1:100; ab108443, Abcam), rabbit anti-motilin (1:50; in-house developed antibody) or rabbit anti-Notch1 (1:100; D1E11, Cell signaling Technology). After washing, enteroids were stained during 2 h with a secondary antibody CY3 donkey anti-goat (1:800; 715-165-150, Jackson ImmunoResearch) or CY5 donkey anti-rabbit (1:800; 711-175-152, Jackson ImmunoResearch), depending upon the host of the primary antibody. Nuclei were stained with DAPI (2.5 μg/ml, ThermoFisher Scientific). For the negative control, the primary antibody was replaced with normal rabbit serum (Jackson ImmunoResearch). Enteroids were mounted back in a 96- CELLSTAR®-plate (Greiner) with Mowiol® 4-88 (Sigma-Aldrich) and visualized using the Operetta CLS High-Content Imaging system (Perkin Elmer). Counting was performed on at least 3-4 views from ± 10 enteroids per patient.
3
Immunofluorescence Staining of Dog Serum Antibodies in J3T Spheroids
4
Immunofluorescence and Proximity Ligation Assay Protocol
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HeLa cells were fixed with 100% methanol for EB1 immunostaining and PLA experiments (anti-EB1 antibody). All other immunostaining and PLA experiments were performed after cofixing cells with 4% paraformaldehyde, PHEM (60 mM Pipes, 25 mM Hepes, 10 mM EGTA, and 4 mM MgCl2, pH 6.9), and 0.5% Triton X-100 for 20 min. Immunofluorescence staining was performed in 3% BSA/TBS–0.05% Tween 20 using these antibodies anti-tubulin DM1-α, antiphospho-H3Ser10 (EMD Millipore), antiphospho–histone H3T3 (EMD Millipore), antiphospho-H2AT120 (Active Motif), phospho-KNL1 (S24 and S60; I.M. Cheeseman, Whitehead Institute for Biomedical Research, Cambridge, MA; Welburn et al., 2010 (link)), anti-AIM1 (Aurora B), anticentromere antigen (Antibodies, Inc.), anti-hEB1, and TO-PRO-3 (Invitrogen).
After the PLA procedure, cells were incubated in 10% normal mouse serum and 10% normal rabbit serum (Jackson ImmunoResearch Laboratories, Inc.) in 3% BSA/TBS + 0.05% Tween 20 for 30 min at RT to block any open binding sites on the PLA probes. FITC-conjugated anti–α-tubulin, DM1-α, and Alexa Fluor 647–conjugated polyclonal antibodies (xlNdc80, xlINCENP, or hBorealin) were used for costaining with PLA reactions. Alexa Fluor 647 polyclonal antibody conjugations were prepared using an Alexa Fluor 647 labeling kit following the manufacturer’s recommended protocol (Invitrogen).
After the PLA procedure, cells were incubated in 10% normal mouse serum and 10% normal rabbit serum (Jackson ImmunoResearch Laboratories, Inc.) in 3% BSA/TBS + 0.05% Tween 20 for 30 min at RT to block any open binding sites on the PLA probes. FITC-conjugated anti–α-tubulin, DM1-α, and Alexa Fluor 647–conjugated polyclonal antibodies (xlNdc80, xlINCENP, or hBorealin) were used for costaining with PLA reactions. Alexa Fluor 647 polyclonal antibody conjugations were prepared using an Alexa Fluor 647 labeling kit following the manufacturer’s recommended protocol (Invitrogen).
5
Immunohistochemistry for SAA in Atherosclerosis
6
Immunofluorescence Staining of Cell Markers
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Paraformaldehyde fixed (4%), permeabilized with 0.1% Triton™ X-100 for 15 min, cells were blocked using 10% normal rabbit serum (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA). Incubation with primary antibodies: anti-TPM3 in concentration 3 µg/mL, MST1 (10 µg/mL), CD276 (1 µg/mL), CD320 (1 µg/mL) diluted in 0.1% BSA, was performed overnight at 4 °C. Afterward, slides were washed in TBS buffer (50 mM Tris-HCl, pH 7.4; 150 mM NaCl) and incubated for one hour with secondary antibodies with Alexa Fluor 594 (0.4 µg/mL, Thermo Scientific). Nuclei were stained with ProLong™ Diamond Antifade Mountant with DAPI (Thermo Fisher). The images were captured at 60X magnification using a confocal microscope, Zeiss LSM710.
7
Immunocytochemistry of Cell Adhesion Molecules
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HEK 293 cells were cultured O/N at 37°C in serum‐supplemented Dulbecco's modified Eagle medium (Lonza, Switzerland) in culture dishes with poly‐D‐coated (Sigma) coverslips. Mammalian expression vectors encoding human CNTN1, CASPR1, CASPR2, NF140, NF155, NF186, NCAM1, and L1CAM (additional information in Table S1 ) in Opti‐MEM (Gibco BRL) were transfected with Lipofectamine 2000 (Invitrogen). Cells were then fixed with 4% PFA (Affymetrix Inc) and blocked for 1 h with either 5% goat serum (Gibco BRL) or 1/40 normal rabbit serum (Jackson Immunoresearch, PA, USA) in PBS. ICC experiments were performed as described in Table S1 . Coverslips were mounted as described above.
8
Murine Skin Single-Cell Preparation and ADAM17 Quantification
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The skin was prepared for flow cytometry as previously described (72 (link), 73 ). Murine ear skin was excised and finely minced and digested with dispase (Thermo Fisher Scientific), type II collagenase (Worthington) and DNAseI (Sigma Aldrich). Cells were then triturated with Pasteur pipettes and further incubated with EDTA before being passed through a 70 μm nylon filter to generate a single cell suspension used for flow cytometric staining.
Skin cells were stained with Fc-block (Biolegend) followed by anti-CD45 anti-EpCAM (all Biolegend) and anti-ADAM17. Donkey anti-rabbit IgG (Jackson Immunoresearch) was used to detect the anti-ADAM17. 4’,6-diamidino-2-phenylindole dihydrochloride (DAPI) was used to exclude dead cells and debris (Sigma Aldrich). Normal rabbit serum (Jackson Immunoresearch) was used as a negative control for ADAM17.
For ADAM17 mean fluorescence intensity (MFI) quantification, all ADAM17 MFIs were normalized to the wild-type control in the same experiment.
Skin cells were stained with Fc-block (Biolegend) followed by anti-CD45 anti-EpCAM (all Biolegend) and anti-ADAM17. Donkey anti-rabbit IgG (Jackson Immunoresearch) was used to detect the anti-ADAM17. 4’,6-diamidino-2-phenylindole dihydrochloride (DAPI) was used to exclude dead cells and debris (Sigma Aldrich). Normal rabbit serum (Jackson Immunoresearch) was used as a negative control for ADAM17.
For ADAM17 mean fluorescence intensity (MFI) quantification, all ADAM17 MFIs were normalized to the wild-type control in the same experiment.
9
Immunofluorescence Staining of 3D Spheroids
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J3T spheroid cells were fixed for 30 minutes with 4% paraformaldehyde (Thermo Fisher Scientific) then embedded in optimal cutting temperature embedding medium as described previously (23 ). Sectioned spheroids were blocked with 5% normal rabbit serum (Jackson Laboratories) in immunofluorescence buffer (0.2% Triton X-100, 0.1% BSA, 0.05% Tween 20 in PBS). Trial dog serum obtained 4 weeks after enrollment was then diluted 1:500 in immunofluorescence buffer and incubated overnight at 4°C. The following day, a rabbit anti-dog IgG H&L-FITC antibody (Jackson Laboratories) was diluted 1:200 and added for 1 hour at room temperature. Background fluorescence was quenched with a 30-minute incubation in 10 mmol/L copper sulfate + 50 mmol/L ammonium acetate solution then counter stained with DAPI (1 μg/mL; Sigma-Aldrich). After immunolabeling, slides were visualized using an Olympus IX3 confocal microscope and processed using Olympus cellSens software.
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