Zenon mouse igg labeling kit

Flow cytometry: The phenotype of freshly isolated and cultured HDMEC and/or BEC and LECs and immune cells was determined by flow cytometry analysis. Cells (5 × 10⁵) were incubated for 30 min at 4 °C in with primary antibodies and live/dead Zombie Aqua (Table 1). Parallel stainings using isotype-matched control antibodies were conducted (Table 1):
Following the incubation with antibodies, cells were washed twice with FACS buffer (0.5% human serum albumin, 0.5 mM EDTA in PBS) and then analyzed by flow cytometry on a FACS ARIA III 4L (BD Biosciences, Allschwil, Switzerland).
Immunohistochemistry: Immunofluorescence staining on cryosections was performed as described before [6 (link),8 (link)] (Table 2).
For double immunofluorescence, some of the primary antibodies were pre-labeled with Alexa 488, 647, or 555-conjugated polyclonal goat F(ab′)2 fragments, according to the manufacturer’s instructions (Zenon Mouse IgG Labeling Kit, Molecular Probes, Invitrogen).

HEK293 and HEK293 EBV-bacterial artificial chromosome (BAC) BALF5Δ cells8 (link) were maintained in Dulbecco’s modified Eagle’s medium (Sigma) supplemented with 10% fetal bovine serum. HEK293EBV-BAC BALF5Δ cells were maintained with addition of 150 μl/ml of hygromycin B to the culture medium. Rabbit anti-BZLF1, -BMRF1, -BALF2, and -BALF5 antibodies were as reported previously7 (link)21 (link). Anti-EBV EA-D-p52/50 mouse monoclonal antibody (MAB8186) was purchased from Chemicon. Anti-FLAG (M2), Anti-α/β-tubulin (#2148) and Anti-HA (6E2) antibodies were from Sigma and Cell Signaling, respectively. Horseradish peroxidase (HRP)-linked goat antibodies to rabbit IgG were from Amersham Biosciences. Secondary goat anti-rabbit, anti-mouse, and anti-rat IgG antibodies conjugated with Alexa 488, 594, 680, and a Zenon mouse IgG labeling kit were obtained from Molecular Probes.

Cells were fixed with 4% paraformaldehyde and processed for indirect immunofluorescence staining, which was conducted using a primary antibody against Flag (Sigma), BBLF1 (Chiu et al., 2012 (link)), or BGLF2, following incubation with fluorescently labeled secondary antibodies (Invitrogen). Mouse anti-BGLF2 monoclonal antibody was generated using the synthesized peptide ₁₄₅KTVEELQDITPS₁₅₅ (Abmart, China). To colocalize BGLF2 with EA-D or gp350, cells were first stained with anti-BGLF2 mAb, then incubated with fluorescently labeled secondary antibody, and finally incubated with fluorescently labeled anti-EA-D (Millipore) or anti-gp350 (Millipore) that had been labeled using a Zenon mouse IgG labeling kit (Invitrogen). Images were captured using a Leica SP5 fluorescence microscope.

For flow cytometric analyses, EBs were dissociated overnight in 1 mg/ml collagenase B (Roche) at 37°C, followed by incubation in TrypLE (Invitrogen) the next morning for 10–15 min to break up remaining EBs. To stain total cardiomyocytes, cells were stained with 1∶500 anti-human SIRPα-PE/Cy7 (BioLegend) and 1∶250 anti-human CD90-FITC (BD Pharmingen) for 1 h at 4°C in PBS/10% FBS staining buffer. Cells were filtered through a 40-µm cell strainer (Fisher) and resuspended at 10⁶ cells/mL in staining buffer for cell sorting. Sorting was performed on an AriaII cell sorter (BD Biosciences). Flow cytometric gates were set using control cells stained with the appropriate isotype control antibody. To determine cardiomyocyte purity, dissociated single cells were fixed with 4% PFA for 15 min at room temperature. Cells were then blocked in 2% BSA, 2% FBS, and 0.01% Triton for 1 h at room temperature. The primary antibody mouse-anti-human cTNT (ThermoScientific, clone 13–11) was conjugated to AlexaFluor 488 invitro using the Zenon Mouse IgG Labeling Kit (Invitrogen), according to manufacturer’s instructions. Conjugated primary antibody was added to blocking solution at 1∶100 final dilution of cTNT antibody for 2 h at room temperature. Cells were analyzed on an LSR-II (BD Biosciences). Data were analyzed using FlowJo software, Version 9.3.2.

Labeling of an anti-pimo antibody with Alexa Fluor dye was performed using Zenon Mouse IgG Labeling Kits (Life Technologies), according to manufacturer’s recommended procedure.