We collected the culture progeny of MuSCs from aged Myf5nLacZ/+/Luciferase double-transgenic mice23 (link),43 (link) by incubation with 0.1% trypsin in PBS for 2 min at 37 °C and transplanted them into tibialis anterior muscles of hindlimb-irradiated NOD/SCID mice. One month after transplant, we injected notexin to damage recipient muscles and activate MuSCs in vivo. Four days later, we collected, fixed, and cryosectioned recipient muscles, as described above. We performed immunohistological analysis of transverse tissue sections to detect β-galactosidase+ cells (indicating a donor-derived cell expressing Myf5, a marker of MuSC activation) in the satellite cell position within the myofiber basal lamina, as defined by laminin staining. We stained sections with anti-Laminin (Millipore, clone A5, catalog # 05-206, 1:250) and anti-β-galactosidase (Invitrogen, catalog # A11132, 1:100) primary antibodies and then with appropriate secondary antibodies (Invitrogen). We counter-stained nuclei with Hoechst 33342 (Invitrogen). We acquired images with an AxioPlan2 epi-fluorescent microscope (Carl Zeiss) with Plan NeoFluar 10×/0.30NA or 20×/0.75NA objectives (Carl Zeiss) and an ORCA-ER digital camera (Hamamatsu). We captured digital images in OpenLab software (Improvision) and assembled them using Photoshop software (Adobe) with consistent contrast adjustments across all images.
Appropriate secondary antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States
Appropriate secondary antibodies are laboratory reagents designed to detect and bind to primary antibodies used in various immunoassays and other immunological techniques. They function as detection tools, providing a signal that indicates the presence and location of the target analyte.
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Lab products found in correlation
Hoechst 33342, by Thermo Fisher Scientific (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (3 mentions)
Openlab software, by PerkinElmer (2 mentions)
Anti laminin, by Merck Group (2 mentions)
Anti β galactosidase, by Thermo Fisher Scientific (2 mentions)
Axioplan2 epifluorescent microscope, by Zeiss (2 mentions)
Orca er digital camera, by Hamamatsu Photonics (2 mentions)
Lsm 510 laser scanning microscope, by Zeiss (2 mentions)
Prolong gold antifade mountant, by Thermo Fisher Scientific (2 mentions)
β actin, by Thermo Fisher Scientific (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Image lab software, by Bio-Rad (2 mentions)
Chemidoc xrs system, by Bio-Rad (2 mentions)
Lsm 700 confocal microscope, by Zeiss (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Ecl plus, by Merck Group (2 mentions)
Rack1, by Santa Cruz Biotechnology (2 mentions)
Rabbit anti zo 1, by Thermo Fisher Scientific (2 mentions)
Lsm 510, by Zeiss (2 mentions)
32 protocols using appropriate secondary antibody
1
Quantifying Activated MuSCs in Transplanted Muscle
2
FCV Infection Visualization in CrFK Cells
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CrFK cells were grown overnight on glass coverslips and infected with FCV at a multiplicity of infection (MOI) of 5 at the indicated times, or at an MOI of 10 for 30 min at 4 °C as indicated. The cells were treated with cytoskeleton buffer for 5 min and permeabilized in 4% formaldehyde solution for 5 min at room temperature (RT). The samples were washed three times with phosphate buffer saline (PBS) for 5 min, blocked with 0.5% gelatin in PBS for 40 min at RT, washed three times with PBS for 5 min, and incubated with the anti-FCV (FCV1-43, Santa Cruz Biotechnology, CA, USA) that recognizes an epitope on the capsid protein or the anti-JAM-1 (ab106114, Abcam, Cambridge, UK), at 4 °C overnight. Samples were washed three times with cold PBS for 5 min and incubated with the appropriate secondary antibodies (Invitrogen, MA, USA) for 1 h at RT. The samples were washed three times with PBS and incubated with 1 mg/mL of 4′6′-diamidino-2-phenylindole (DAPI) for 2 min. The samples were washed six times with PBS and three times with distilled water. The samples were treated with VECTASHIELD liquid mounting media (Vector Laboratories A.C., CA, USA) and analyzed using a Zeiss LSM-700 confocal microscope.
3
Immunofluorescence Confocal Microscopy Protocol
4
Western Blotting of METTL14, CSNK1D, and SLC35E1
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Western blotting was conducted as previously described (22 (link)). Briefly, protein concentrations were measured with a BCA Kit. Protein lysates were resolved using SDS–PAGE and transferred onto PVDF membranes (Millipore). The membrane was subsequently incubated overnight (4°C) with the following primary antibodies: anti-METTL14 (Norvus), anti-CSNK1D (Norvus), anti-SLC35E1 (Norvus) and β-actin (Invitrogen). After washing, the membranes were further subjected to the appropriate secondary antibodies (Invitrogen). Blots were visualized by a ChemiDoc XRS system, followed by quantification using Image Lab software (Bio–Rad).
5
Immunocytochemical Analysis of Apoptosis
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Cells cultured on coverslips were fixed with 4% paraformaldehyde at 4 °C for 30 min. After washing with PBS, cells were permeabilized in PBS containing 1% Triton X-100 and 0.5% NP-40 (Sigma-Aldrich) and then blocked with 3% bovine serum albumin (BSA) and 1% normal horse serum (Santa Cruz Biotechnology). Cells were incubated with antibodies against Tuj1 (1:100, Abcam, Cambridge, UK), cleaved caspase3 (1:100, Cell Signaling Technology), and then appropriate secondary antibodies (Invitrogen). Cells were stained with a TUNEL kit (Roche, Indianapolis, IN, USA), according to the manufacturer’s protocol. 4′,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA, USA) was used for the nuclei. After mounting, the cells were examined using confocal laser scanning microscopy (BIORP, Leica, Wetzlar, Germany).
6
Immunofluorescence of CIP4 and N-WASP
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H1299 cells growing on glass coverslips coated with 10 μg/ml fibronectin (Roche) were fixed (4% PFA), permeabilized (0.2% Triton-X100), and incubated in blocking buffer (5% goat serum, 5% BSA) for 30 min. Primary antibodies (mouse α-CIP4, 1:100; rabbit α-N-WASP, 1:100) were detected with appropriate secondary antibodies (Invitrogen). Images were acquired by confocal microscopy.
7
Immunohistochemical and Confocal Imaging Protocol
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Tissue samples embedded in paraffin blocks were obtained from the Biological Resource Centre of the Pathology Department, Centre Antoine Lacassagne, Nice. Informed written consent was obtained from all the patients in accordance with institutional guidelines and the experimental protocol, that complied with ethical and safety practices for research involving human tissues, was approved by the CPP Sud-Méditerranée V ethics committee (National Authorisation Number: AC-2014-2237). Immuno-staining was performed as described40 (link). For fluorescence, slides were labelled with appropriate secondary antibodies (Invitrogen) followed by nuclear staining with DRAQ5 (Thermo Scientific) and then mounted in ProLong Gold antifade reagent (Invitrogen). Immunohistochemically stained sections were digitalized using a Hamamatsu NanoZoomer 2.0-HT Digital slide scanner (40X mode). Images were visualized and captured using NDP.view2:U12388-01 software. Fluorescently-stained sections were analysed and images acquired with a 10x/0.45 or 40x/1.1 W objective on a Zeiss NLO780 confocal system.
8
Characterizing Stem Cell Surface Markers
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hWJSCs and hBMMSCs were cultured in a Lab-Tek® Chambered #1.0 Borosilicate coverglass system (Thermo Scientific) until confluence. The cells were then fixed in 4% paraformaldehyde for 10 min, permeabilized with 0.1% Triton-X100 for 10 min, and then washed with PBS. Non-specific blocking was carried out with 10% normal goat serum for 10 min. The cells were incubated with primary antibodies CD29, CD44, CD146, VCAM-1, MMP-2, MMP-9, SDF-1a, ICAM-1, fibronectin, laminin, hyaluronic acid, and collagen type IV (Biolegend, San Diego, USA) at 4 °C overnight. The cells were then incubated with appropriate secondary antibodies (Invitrogen Life Technologies) for 30 min at room temperature in the presence of 1 mg/mL Hoechst 33342 and mounted on to slides with appropriate mounting medium. Photographs were taken using an Olympus FluoView FV1000 laser scanning confocal microscope (Olympus, Japan).
9
Quantifying Cardiac Fibrosis and Inflammation
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The hearts were harvested and fixed with 4% paraformaldehyde and embedded in paraffin. The sections were then immunostained with the rabbit anti–green fluorescent protein (GFP) antibody (GeneTex) and rabbit anti–β‐Gal antibody (Invitrogen). For visualization, a diaminobenzidine substrate kit (Vector Laboratories) was used. To evaluate the degree of fibrosis, Masson's trichrome staining was performed and the infarct size was quantified using the midline length measurement, as previously reported.19 In brief, infarct size was evaluated from 3 sections per sample. To measure the infarct size, the sum of midline infarct lengths from all sections was divided by the sum of midline circumferences from all sections. The infarct size value is presented as a percentage. For immunofluorescent staining of macrophages, the collected hearts were processed for frozen sectioning by embedding in the OCT media. The sections were stained with the mouse anti‐cTnT (DSHB) and rat anti‐F4/80 (Abcam). Appropriate secondary antibodies (Invitrogen) were used for visualization under a fluorescence microscope. Nuclei were stained with 4,6‐diamidino‐2‐phenylindole (1 μg/mL; Sigma) for visualization.
10
Immunofluorescence Confocal Microscopy Protocol
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