After deparaffinization and rehydration, sections were incubated in citrate buffer for 16 h at 60°C to retrieve the antigen. Sections for immunohistochemistry (IHC) were treated with 3% hydrogen peroxide for 15 min and then blocked with 1% goat serum for 1 h. Then sections were incubated with primary antibodies against CRLF1 (1:100, ab211438, Abcam), GFP (1:100, EPR14104, Abcam), and a-SMA (1:100, ab7817, Abcam) for IHC, and a-SMA (1:100, ab7817, Abcam), CRLF1 (1:100, ab211438, Abcam) for Immunofluorescence (IF). Species-matched secondary antibody was used. Then, DAB (Beyotime, China) and haematoxylin (Beyotime, China) were used for IHC, and DAPI was used for IF.
Epr14104
Manufactured by Abcam
Sourced in United Kingdom
EPR14104 is a recombinant antibody specific for the Histone H2A.X (phospho S139) protein. It is designed for use in applications such as Western Blotting, Immunohistochemistry, and Immunocytochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Haematoxylin, by Beyotime (1 mentions)
Ab211438, by Abcam (1 mentions)
3 3 diaminobenzidine (dab), by Beyotime (1 mentions)
Ab7817, by Abcam (1 mentions)
Anti rfp 5f8, by Proteintech (1 mentions)
Anti gfp 4b10, by Cell Signaling Technology (1 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
Gl 7 clone gl 7, by Thermo Fisher Scientific (1 mentions)
Cd21 35 clone ebio4e3, by Thermo Fisher Scientific (1 mentions)
Imaris software 8, by Oxford Instruments (1 mentions)
Cryostat, by Leica (1 mentions)
Optimal cutting temperature medium, by Electron Microscopy Sciences (1 mentions)
B220 clone ra3 6b2, by BioLegend (1 mentions)
Alexa fluor 488 goat anti chicken, by Thermo Fisher Scientific (1 mentions)
Er tr7, by Abcam (1 mentions)
Ab172958, by Abcam (1 mentions)
Glutathione sepharose, by GE Healthcare (1 mentions)
Mouse anti halotag, by Promega (1 mentions)
Ab167453, by Abcam (1 mentions)
5 protocols using epr14104
1
Immunohistochemical and Immunofluorescence Analysis
2
Histological and Immunohistochemical Analysis of Tissue Samples
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All tissue samples were fixed with 4% paraformaldehyde overnight, decalcified with pH 7.2 EDTA buffer (G-Chelate Mild, GenoStuff, Tokyo, Japan) for 10 d at 4 °C, and embedded in paraffin. The samples were cut into 3–4-μm-thick sections. Hematoxylin and eosin, safranin O/fast Green, and Masson trichrome staining were applied using standard protocols. For immunohistochemistry, the antibodies used were anti-GFP (EPR14104) (Abcam; dilution 1:100), anti-GFP (4B10) (Cell Signaling Technology; dilution 1:200), anti-RFP (5F8) (Chromotek; dilution 1:200), anti-tenomodulin (Thermo Fisher Scientific; dilution 1:150), anti-αSMA (1A4) (Abcam; dilution 1:200), and anti-bFGF (Bioss; dilution 1:200). For DAB staining, the secondary antibody used was Dako EnVision (Dako Japan Inc., Kyoto, Japan); stained cells were analysed by microscopy (BX51, Olympus). For immunofluorescence, the secondary antibodies were conjugated with Alexa Fluor 488 and Alexa Fluor 594 (Thermo Fisher Scientific); stained cells were analysed by fluorescence microscopy (IX83, Olympus).
3
Confocal Microscopy of Infected Spleen Sections
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For confocal microscopy of frozen spleen sections, spleens were removed at the indicated day post-infection, fixed in periodate-lysine-paraformaldehyde fixative for 48 h, and then moved to 30% sucrose/PBS solution for 24 h. Tissues were embedded in optimal-cutting-temperature medium (Electron Microscopy Sciences) and frozen in dry-ice-cooled isopentane. 16-µm sections were cut on a Leica cryostat (Leica Microsystems, Buffalo Grove, IL). Sections were blocked with 5% goat, donkey, bovine, rat, or rabbit serum and then stained with a combination of the following Abs: ERTR7 (Abcam, Boston, MA), B220 (clone RA3-6B2, Biolegend), GL7 (clone GL-7, Thermo Fisher Scientific), and CD21/35 (clone eBio4E3, eBioscience, San Diego, CA). Sections were incubated with secondary antibodies as needed and for controls, and images were acquired on a Leica SP8 or Stellaris Confocal microscope. To increase staining intensity of MHV68, spleens were stained with anti-GFP Ab (EPR14104, Abcam) and goat anti-chicken Alexa Fluor 488 (ThermoFisher Scientific, Waltham, MA). Images were processed and analyzed using Imaris software 8.0 (Oxford Instruments). Where indicated, the spots function of Imaris was used to identify and create spots for MHV68+ cells. Spots were masked on TdTomato expression inside the spot to reveal MHV68+ tdTomato+ cells, referred to as “gated.”
4
Antibody Production and Characterization
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The following antibodies were used in this study: rabbit anti-CHC17 (one made in house and one purchased from ab172958; Abcam); mouse anti-CHC17 (X22; Abcam); rabbit anti-AP1G1 (made in house); rabbit anti-GFP (one a gift from Matthew Seaman, CIMR, Cambridge, UK, and one purchased from EPR14104; Abcam); mouse anti-HaloTag (G9211; Promega); rabbit anti-CIMPR (made in house); mouse anti-AP-2 α subunits (AC1-M11; Abcam); and mouse anti-GGA2 (a gift from Doug Brooks, University of South Australia, Australia).
In addition, a new antiserum was produced against mRuby2, which was not recognized by antibodies against other red fluorescent proteins because it comes from a different organism (Entacmaea quadricolor instead of Discosoma). The entire coding sequence was expressed as a GST fusion protein, affinity-purified using glutathione-Sepharose (GE Healthcare), and used to raise antibodies in rabbits following our established protocol (Page et al., 1999 (link)). The titer of the serum was found to be extremely high, producing very strong labeling in both immunofluorescence and Western blotting experiments when it was diluted 1:10,000, so it was not affinity purified.
In addition, a new antiserum was produced against mRuby2, which was not recognized by antibodies against other red fluorescent proteins because it comes from a different organism (Entacmaea quadricolor instead of Discosoma). The entire coding sequence was expressed as a GST fusion protein, affinity-purified using glutathione-Sepharose (GE Healthcare), and used to raise antibodies in rabbits following our established protocol (Page et al., 1999 (link)). The titer of the serum was found to be extremely high, producing very strong labeling in both immunofluorescence and Western blotting experiments when it was diluted 1:10,000, so it was not affinity purified.
5
Western Blot Detection of Fluorescent Proteins
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Rabbit monoclonal antibody against GFP (EPR14104, ab183734), rabbit polyclonal antibody against mCherry (ab167453), and rabbit polyclonal to HIV-1 gag (p55 + p24 + p17, ab63917) were purchased from Abcam (UK). Horse-radish peroxidase-conjugated goat anti-rabbit was obtained from Agilent Dako (USA). NuPAGE™ 4-12% Bis-Tris Protein Gels and iBlot™ Transfer Stacks were purchased from ThermoFisher Scientific (USA). Immunoblotted proteins were detected using the Enhanced Chemiluminescence detection system (Amersham, UK) and ChemiDoc XRS+ (Bio-Rad, USA). Pan-caspase inhibitor zVAD-FMK was purchased from Enzo Life Sciences (USA) and GrM inhibitor AcKVPL-CMK from Peptanova (Germany).
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