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Polyclonal rabbit anti human igg

Manufactured by Agilent Technologies
Sourced in Denmark

Polyclonal rabbit anti-human IgG is a laboratory reagent used for the detection and quantification of human immunoglobulin G (IgG) in biological samples. It consists of a mixture of antibodies derived from rabbits that have been immunized with human IgG, allowing for the recognition and binding of various IgG epitopes.

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5 protocols using polyclonal rabbit anti human igg

1

Flow Cytometry Staining Protocol

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All antibodies were used FITC, PE, Cy3, PE-Cy5, PE-Cy7, APC, eFluor660 or biotin conjugated. The biotinylated mAb were revealed with Streptavidin-PE-Cy5. mAb were used at 0.1–1 µg/1×106 cells in 100 µl PBS/2% FCS/0.02% NaN3 (FACS buffer) or HBSS/0.3% BSA incubated on ice for 20–30 min. Antibodies used were: anti-CD16/CD32 (Fcγ III/II Receptor) (2.4G2), anti-CD90.1 (Thy1.1) (HIS51), anti-CD8α(Ly-2) (53–6.7), anti-CD3ε (145-2C11), anti-Vα2 T cell receptor (B20.1), anti-α4β7 (DATK32), anti-IAb (AF6-120.1) and anti-CD11c (HL3) all from BD Biosciences (Heidelberg, Germany), anti-CCR9 (B cell hybridoma supernatant, kindly provided by Prof. R. Förster), anti-rat IgG (secondary for CCR9) (from Jackson ImmunoResearch Europe Ltd.), anti-CCR10 (248918), E-selectin/human IgG-Fc-Chimera (both from R&D) and polyclonal rabbit anti-human IgG (secondary for E-lig staining) (DakoCytomation, Hamburg, Germany).
All stainings were performed following a standard protocol except of E-lig staining. This staining was performed as described [16] (link). Control staining for E-lig and CCR9 was done with secondary Ab only, while corresponding isotype control mAb or fluorescence minus one (FMOs) were used as a control for all other stainings.
Data were acquired and analysed on a FACScan using CellQuestPro software (BD Biosciences) or on a FACS Canto II (Becton Dickinson) and analysed using FlowJo.
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2

ELISA Protocol for AE and CE Antibody Detection

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All ELISAs were performed in principle as described by Schweiger et al., (2012) [20 (link)] and the antigens were coated according to previous titration studies [20 (link)]. In this study, an anti-human-immunoglobulin G (IgG; polyclonal rabbit anti-Human-IgG, specific for the Fc part of the heavy chain; DAKO, Glostrup, Denmark) conjugated to alkaline phosphatase (Roche Applied Science, Rotkreuz, Switzerland) was used with a dilution of 1:500. Positive standard sera of human patients with confirmed AE or CE and negative standard sera of blood donors were included in all test runs and ELISA plates (5 sera per plate).
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3

Quantification of Immunoglobulin G and Interleukin-6 in Cell Cultures

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IgG levels in culture supernatants were detected by ELISA. Briefly, microtiter plates (Greiner, Milan, Italy) were coated with polyclonal rabbit anti-human IgG (Dako, St. Clara, CA, USA) and incubated 3 h at 37 °C and then overnight at 4 °C. After washes, culture supernatants were incubated for 2 h at 37 °C and horse-radish peroxidase (HRP)-conjugated rabbit anti human IgG (Dako) was added. After 2 h of incubation, substrate solution (o-Phenylenediamine dihydrochloride) (Sigma Aldrich) was added. Colorimetric reaction was read at 492 nm (Sunrise, Tecan, Switzerland). Data were calculated by Magellan Software (Tecan). IgG levels were expressed as µg/mL.
IL-6 levels were detected by ELISA using Duo set antibody pairs (R&D System, Minneapolis, MN, USA) according to the manufacturer’s instructions. Results were expressed as pg/mL.
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4

EBV Lytic Cycle and Autophagy Monitoring

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HeLa cells and the EBV-positive Burkitt’s lymphoma cell line Akata (purchased from the American Type Culture Collection (ATCC)) were cultured at 37 °C under 5% CO2 in RPMI-1640 (Gibco, Illkirch, France) supplemented with 10% fetal calf serum (FCS; Thermo Scientific, Illkirch, France). HeLa cells with stable expression of GFP-LC3 and mRFP-GFP-LC3 were kindly provided by Aviva Tolkovsky [38 (link)] and David Rubinsztein [39 (link)], respectively. These cells were grown in RPMI-1640 supplemented with 10% FCS and G418 (500 µg/mL; Invivogen, Toulouse, France). In order to induce the EBV lytic cycle, Akata cells were treated with 7.5 µg/mL of polyclonal rabbit anti-human IgG (Dako, Santa Clara, USA). Autophagic flux was monitored by the addition of chloroquine (50 μM) 4 h prior to cell lysis. Starvation-induced autophagy was carried out by replacing the complete medium with Earle’s Balanced Salt Solution (EBSS; Gibco) for 4 h before performing immunofluorescence staining. DNA plasmid transfections were performed by using Fugene HD transfection reagent (Promega, Madison, USA) according to the manufacturer’s instructions.
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5

Histological Analysis of Excised Kidney Tissue

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Excised GalT-KO kidney tissue samples were divided into three sections. Two were histologic examination and one was for CMV analysis. Formaldehyde fixed tissue samples were stained using either hematoxylin and eosin (H&E) and periodic acid-Schiff (PAS) stains. Coded samples were examined by light microscopy, and rejection was diagnosed according to a standardized grading system (25 (link)). Immunohistochemistry was also performed on frozen sections using polyclonal antibodies reactive to ICAM-1, porcine MHC Class II, and IgG (polyclonal rabbit anti-human IgG (Dako, Denmark).
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