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3 protocols using epha2

1

Antibody Staining and Flow Cytometric Analysis

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Cells were detached with Versene (Invitrogen) counted and resuspended in 100 µL phosphate buffered saline (PBS) containing 1% bovine serum albumin (BSA). Cells were blocked with PBS/1% BSA for 1 h on ice. Two micrograms of QUAD 3.0, EphA3 antibody (house made),9 (link) EphA2, EphB2 (R&D Systems), or IL-13RA2 (Biolegend) was added and incubated for 2 h on ice with occasional mixing. Cells incubated with isotype antibody served as controls. Cells were washed with PBS/1% BSA prior to the addition of Alexa-Fluor-labeled secondary antibodies. Cells were incubated on ice for an additional 1 h with occasional mixing. After washing with PBS/1% BSA, cells were postfixed with 10% buffered formalin. Cells were analyzed on an Accuri 6 flow cytometer. Data were analyzed using the FCS Express software (DeNovo Software).
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2

Immunohistochemical Analysis of Mouse Eye Tissue

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Isolated mouse eyes were fixed in 3.7% formaldehyde overnight at 4 °C, cryoprotected in 30% sucrose solution for a minimum of 24 h prior to freezing and 20-μm thick cryosections cut. Sections were incubated in 0.25% Triton X-100 in DPBS buffer (2.7 mM KCl, 1.5 mM KH2PO4, 137.9 mM NaCl, 8.1 mM Na2HPO4–7 H2O [Corning, 21-0310CV]) for 10 min, followed by blocking buffer (5% goat serum, 0.5 g BSA in 50 ml DPBS) for 1 h prior to labeling. Samples were incubated sequentially in primary antibody at 4 °C overnight, followed by fluorescent-conjugated secondary antibody for 1 h at 37°C (Jackson ImmunoResearch Laboratories, 111-295-144, 115-545-003, 115-295-008). Primary antibodies used included: N-cadherin (BD Bioscience, 610921), β-catenin (BD Bioscience, 610154), E-cadherin (Cell Signaling, 24E10), βB-crystallin (Santa Cruz, FL-252), Rac1 (Abcam, ab155938), myosin-II (LifeSpan BioSciences, LS-C84042), WGA (LifeSpan BioSciences, LS-C76576), ZO-1 (Abcam, 61357), EphA2 (R&D, AF639), connexin 50 (ADI, Cx50-A), aquaporin-0 (ADI, AQP01-A), active Rac1 (NewEast Biosciences, 26903), p-myosin (Cell Signaling, 3674), and MLCK (Abcam, ab76092). F-actin was localized with Alexa448-conjugated phalloidin (Invitrogen-Molecular Probes). Nuclei were labeled with TO-PRO-3 (Invitrogen-Molecular Probes).
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3

Immunophenotyping of γδ T Cells

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The cell phenotype was assessed by fluorescence-activated cell sorting (FACS) by using staining with the monoclonal antibodies MICA, MICB, CD112, CD155, B7-H6 (R&D Systems, clone 875001), and EphA2 (R&D Systems, clone 371805). The tumor cell lines were harvested, washed with cold phosphate-buffered saline (PBS, Sigma) containing 2% FBS, and incubated for 30 min on ice with fluorescently labelled monoclonal antibodies. Gamma-delta T cells were identified in freshly isolated PBMCs labelled with CD3 (Thermo Fisher Scientific, clone SK7), Vδ1 TCR (Thermo Fisher Scientific, clone TS8.2), Vδ2 TCR (BD Pharmingen, clone B6) or Vδ2 TCR (Sony, clone B6). CD27 (BD Pharmingen, clone M-T271), and CD45RA (Exbio, clone MEM-56) were used for immunophenotyping. Samples were washed and acquired using FACSCanto® (BD Biosciences) and data analyzed using FlowJo® software (FlowJo, Ashland, OR, USA). Forward and side scatter gating were used to discriminate live cells from dead cells and γδ T cells were derived from SSC vs. FSC-gated bulk PBMCs with doublet exclusion (FSC-A vs. FCS-H). To determine the placement of the gates, appropriate fluorescence minus one (FMO) and unstained controls were used.
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