As previously described (25 ), asynchronous DMS114 and H524 cells were sequentially labelled with 20 μM IdU for 20 min and 50 μM CldU for 20 min. To preserve long genomic DNA fibers, cells were embedded in low melting point agarose plugs and incubated in cell lysis buffer with proteinase K at 50°C for overnight. Washed plugs with TE buffer, and then melted plugs in 0.1M MES (pH 6.5) at 70°C for 20 min. Agarose was subsequently degraded by adding 2 μl of β-agarase (New England Biolabs). DNA fibers were then stretched onto salinized coverslips (Genomic Vision, cov-002-RUO) using an in-house combing machine. Combed DNA on coverslips was then baked at 60 °C for 2 hours and denatured in 0.5 N NaOH for 20 min. IdU, CldU and single-strand DNA were detected using a mouse antibody directed against BrdU (IgG1, Becton Dickinson, 347580, 1:25 dilution), a rat antibody directed against BrdU (Accurate chemical, OBT0030, 1:200 dilution) and a mouse antibody directed against single-stranded DNA (ssDNA) (IgG 2a, Millipore, MAB3034, 1:100), respectively. The secondary antibodies used were goat anti-mouse Cy3 (Abcam ab6946), goat anti-rat Cy5 (Abcam, ab6565), and goat anti-mouse BV480 (Jackson ImmunoResearch, 115–685-166) for ssDNA. Slides were scanned with a FiberVision Automated Scanner (Genomic Vision). Replication signals on single DNA fibers were analyzed using FiberStudio (Genomic Vision).
Igg2a
Manufactured by Merck Group
Sourced in United States, Macao
IgG2a is a laboratory product marketed by Merck Group. It is a type of immunoglobulin G (IgG) antibody subclass. IgG2a is a key component used in various immunological and biochemical applications.
Automatically generated - may contain errors
Lab products found in correlation
Igg2b, by Merck Group (12 mentions)
Mouse anti ctbp2 antibody, by BD (2 mentions)
Hypoxanthine thymidine medium, by Merck Group (2 mentions)
Rpmi 1640, by Merck Group (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Igg2c, by Southern Biotech (2 mentions)
Ab6946, by Abcam (1 mentions)
β agarase, by New England Biolabs (1 mentions)
Mab3034, by Merck Group (1 mentions)
Glycine, by Avantor (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Triton x 100, by AppliChem (1 mentions)
Superfrost slide, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 goat anti chicken, by Thermo Fisher Scientific (1 mentions)
Tcs lsi confocal microscope, by Leica (1 mentions)
Mowiol, by Polysciences (1 mentions)
Mab302, by Merck Group (1 mentions)
Goat anti mouse igg2b alexa fluor 647, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 647 goat anti mouse igg1, by Thermo Fisher Scientific (1 mentions)
Tissue freezing medium tfmtm, by Electron Microscopy Sciences (1 mentions)
35 protocols using igg2a
1
Measuring Replication Dynamics in Cells
2
Synapses per Inner Hair Cell Quantification
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The immunohistochemistry method to assess the number of synapses per inner hair cell (IHC) has been extensively described [14 (link)]. Briefly, the presynaptic IHC ribbons were identified using a mouse anti-CtBP2 antibody (1:500; BD Biosciences, San Diego, CA). Glutamate receptors were labelled with a mouse antibody raised against the C-terminus of the GluA2 subunit, IgG2a (1:200, Millipore, Billerica, MA). A 3D, custom algorithm was used to detect the juxtaposition of pre- and post-synaptic structures in stacked confocal images.
3
Immunohistochemical Quantification of Synapses per Inner Hair Cell
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The immunohistochemical method for assessing the number of synapses per inner hair cell (IHC) has been described in detail elsewhere (Bourien et al. 2014; Batrel et al. 2017) . Briefly, the presynaptic IHC ribbons were identified with a mouse anti-CtBP2 antibody (1:500; BD Biosciences, San Diego, CA). Glutamate receptors were labeled with a mouse antibody raised against the C-terminus of the GluA2 subunit, IgG2a (1:200, Millipore, Billerica, MA). A 3D, custom algorithm was used to detect the juxtaposition of pre-and post-synaptic structures in stacked confocal images. Once the ribbons had been counted, the corresponding coding frequency of each ribbon was inferred from the rat cochlear place frequency map (Müller 1991) . A second-order polynomial was then fitted to synapse count as a function of position relative to the cochlea apex (Meyer et al. 2009 ).
4
Tricolor Glutamatergic Synapse Visualization
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For the triple staining of glutamatergic synapses on PNN positive neurons, coverslips were washed three times with washing solution containing 10% (v/v) fetal bovine serum (FBS) (Sigma-Aldrich; Cat. No.: F7524), 0.1 mM glycine (VWR International GmbH; Cat. No.: 101196X), and 0.1% (v/v) Triton X-100 (AppliChem; Cat. No.: A4975,0500) in PBS, after fixation. Then, following antibodies were diluted in washing solution: anti-vesicular glutamate transporter-1(vGlut) (1:1,000; guinea pig; polyclonal; Synaptic Systems GmbH; RRID: AB_887878 ), anti-postsynaptic density-95 (PSD-95) (1:500; mouse; monoclonal IgG2a; Merck KGaA; RRID: AB_1121285 ), and anti-aggrecan (1:500; rabbit; polyclonal; Merck KGaA; RRID: 90460 ). Coverslips were incubated for 1 h at room temperature. Then, coverslips were washed three times with washing solution and incubated with the secondary antibody solution for 1 h at room temperature. Here, following antibodies were used: anti-guinea pig (1:500; AF647; IgG (H+L); Jackson Immuno Research, RRID: AB_2337446 ), anti-mouse, (1:250; AF488; IgM+IgG (H+L); Jackson Immuno Research; RRID: AB_2338844 ), and anti-rabbit (1:500; Cy3; IgG (H+L); Jackson Immuno Research; RRID: AB_2338003 ). Last, coverslips were washed three times with PBS (1x), once with MilliQ water and finally mounted on microscope slides.
5
Immunolabeling of EAAT2a in Zebrafish
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Generation of the chicken anti‐EAAT2a (zebrafish) antibody is described elsewhere (Niklaus et al., 2017 (link)). Five dpf larvae were fixed in 4% PFA in phosphate buffered saline (PBS, pH 7.4) at room temperature for 40 min. Embryos were cryo‐protected in 30% sucrose in PBS at 4°C overnight, embedded in Tissue Freezing Medium TFMTM (Electron Microscopy Sciences), cryo‐sectioned at 14–16 μm and mounted onto Superfrost slides (Thermo Fisher Scientific). Immunohistochemistry was performed as described before (Niklaus et al., 2017 (link)). Chicken anti‐EAAT2a 1:500, mouse anti‐synaptic vesicle 2 (IgG1, 1:100, DSHB USA), mouse anti‐acetylated tubulin (IgG2b, 1:500, Sigma 7451) and mouse anti–glutamine synthetase (IgG2a, 1:200, EMD Millipore, MAB302) were used as primary antibodies. Secondary antibodies were goat anti‐chicken Alexa Fluor 488, goat anti‐mouse IgG2a Alexa Fluor 568, goat anti‐mouse IgG2b Alexa Fluor 647 and goat anti‐mouse IgG1 Alexa Fluor 647, all 1:500 (all from Invitrogen, Thermo Fisher Scientific). Slides were cover‐slipped using Mowiol (Polysciences) containing DABCO (Sigma‐Aldrich) and imaged with a TCS LSI confocal microscope (Leica Microsystems). Images were adjusted for brightness and contrast using Affinity Photo Version 1.8 and assembled in Affinity Designer Version 1.7.
6
Murine Anti-HCV IgG Antibody Detection
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To detect the presence of murine anti-HCV IgG antibodies, an endpoint dilution ELISA was carried out as described previously [23 (link)]. Following the same protocol, we also assessed the presence of IgG1 and IgG2a antibody subclasses using horseradish-peroxidase-conjugated goat anti-mouse IgG1 and IgG2a diluted 1:15,000 and 1: 60,000, respectively (Sigma-Aldrich).
7
Quantifying Antibody Isotype Profiles
8
Localization of Pex14, Pex16, and Myo-IDH in Entamoeba Cells
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Transmission electron microscopy was performed using E. histolytica cells overexpressing polyHis- or V5-tagged Pex14 and Pex16, and samples were processed as described previously [27 (link)]. The sections with V5-tagged Pex14 were blocked with 1% fish skin gelatin (FSG) and immunodecorated using mouse anti-V5 antibody (1:20, Invitrogen/Thermo Fisher Scientific, Waltham, MA, USA) and goat anti-mouse antibody conjugated with 5 nm gold nanoparticles (BBI Solutions) for 1 h in 1% fish skin gelatin. Samples with polyHis-tagged Pex14 were blocked in buffer containing 1% bovine serum albumin/0.05% Tween 20 in 0.1 M HEPES for 1 h at room temperature. Labeling was performed using Ni-nitrilotriacetic acid (Ni-NTA) conjugated with 5 nm gold nanoparticles (Nanoprobes, Yaphank, USA). Sections with polyHis-tagged Pex16 were treated as described above or with mouse monoclonal anti-polyHis antibody (IgG2A, Merck/Sigma-Aldrich, St. Louis, MO, USA), and protein A conjugated with 5 nm gold nanoparticles (1:50)(CMC Utrecht) in 1% fish skin gelatin. Myo-IDH was detected using rabbit polyclonal anti-myo-IDH antibody and anti-rabbit IgG conjugated to 15 nm gold particles (Nanoprobes, Yaphank, USA).
9
Serological Assessment of Immunization
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Four weeks after the third injection, the sera of mice were collected and pooled for each group and stored at − 20 °C. The levels of total IgG, IgG1, IgG2a and IgG2b (1:10,000 v/v, Sigma) were assessed by indirect ELISA against the Nef-Vpr-Gp160-P24 polypeptide or Nef protein as an antigen (5 µg/ml). TMB (Tetra Methyl Benzidine, Sigma) was used as a substrate and the absorbance was assessed at 450 nm (Bolhassani et al.2008 (link)).
10
Characterizing Skin Stem Cell Populations
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Samples analyzed were treated with 5 U/ml Dispase (Sigma-Aldrich, Saint Louis, MO, USA) in agreement with the protocol previously published [15 (link)]. Samples were stained with different combinations of antibodies to ITGA6 (BD Biosciences — Pharmingen clone GoH3) and CD200 (MRC OX-104), fixed and permeabilized (Caltag labs), and stained with the anti-human antibodies against actin (Sigma-Aldrich clone AC-15), KRT15, FST, Ki67 (BD Biosciences - Pharmingen clone B56), CD117 (BD Biosciences clone 104D2), CD45 (BD Biosciences clone 2D1), Ber-EP4, DAPI, and/or an isotype control (IgG2a, Sigma-Aldrich). Analyses were realized on a dual-laser flow cytometer (BD FACSCalibur). All flow cytometry data were analyzed by uploading files into FlowJo 4.6 (TreeStar Inc., Ashland, OR, USA) [15 (link)]. KRT15, CD200 and CD34 expression were analyzed to assess the stem and progenitor cell compartments in the targeted area treated with HD-AFSCs at baseline and after treatment.
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