Brain sections mounted on slides were processed for antigen unmasking by using H-3300 Vector Antigen Unmasking Solution. Slides were blocked with blocking solution (3% Normal Rabbit Serum, 0.3% Triton-X 100, in 1x PBS) and incubated overnight with primary antibody anti-CD8α (1:100, Thermofisher #2025-05-31) for staining. Sections were then stained with secondary antibody biotinylated anti-Rat-IgG in 1.5% NRS for 60 mins at room temperature. Treatment with Avidin-biotin complex and DAB peroxidase substrate was used to visualize the staining. 40x digital images of the sections were obtained using whole slide imaging (Nanozoomer 2.0HT, Hamamatsu Photonics, Hamamatsu-shi, Japan) and positive cells were manually quantified using Image J software.
Anti cd8α
Manufactured by Thermo Fisher Scientific
Sourced in Germany, United States
Anti-CD8α is a monoclonal antibody that binds to the CD8α protein, which is expressed on the surface of certain T cells and natural killer cells. This antibody can be used for the identification and enumeration of these cell types in flow cytometry and other immunological applications.
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Lab products found in correlation
Anti cd4, by Thermo Fisher Scientific (6 mentions)
Anti ifn γ, by Thermo Fisher Scientific (3 mentions)
Anti cd3, by Thermo Fisher Scientific (3 mentions)
Anti cd11c, by Thermo Fisher Scientific (3 mentions)
Anti cd45, by Thermo Fisher Scientific (2 mentions)
Anti cd86, by Thermo Fisher Scientific (2 mentions)
Anti f4 80, by Thermo Fisher Scientific (2 mentions)
Anti cd44, by Thermo Fisher Scientific (2 mentions)
Anti cd62l, by Thermo Fisher Scientific (2 mentions)
Lsrfortessa, by BD (2 mentions)
Click it plus edu alexa fluor 647 flow cytometry assay kit, by Thermo Fisher Scientific (2 mentions)
Anti cd8β, by Thermo Fisher Scientific (2 mentions)
Anti vγ1 clone 2, by BioLegend (2 mentions)
Anti cd11b, by Thermo Fisher Scientific (2 mentions)
Nanozoomer 2.0 ht, by Hamamatsu Photonics (1 mentions)
Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (1 mentions)
Anti tnf α, by Thermo Fisher Scientific (1 mentions)
Anti ki67, by Thermo Fisher Scientific (1 mentions)
Fixable viability stain 700, by BD (1 mentions)
Anti cd3e, by Thermo Fisher Scientific (1 mentions)
14 protocols using anti cd8α
1
CD8α Immunohistochemistry Protocol
2
Comprehensive Immune Profiling of Tumor Samples
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Tumors were collected, minced, and digested with collagenase B (0.5 mg/mL, Roche) and hyaluronidase (0.5 mg/mL, Absin) at 37°C for 1 hour. Suspensions were filtered through 40 µm cell strainers. Then, cells were suspended and stained with Fixable Viability Stain 700 (BD Biosciences). Cells were stained according to the standard protocol for flow cytometry. The following antibodies and buffers were used (all reagents from BD Biosciences unless otherwise indicated): anti-CD45 (560510), anti-CD3e (562600), anti-CD8α (563068), anti-CD25 (553075), anti-CD69 (566500), anti-Ki67 (556027), anti-TNF-α (563943), anti-IFN-γ (560660), anti-Perforin (ThermoFisher, 11-9392-82), anti-Granzyme-B (BioLegend, 372204), anti-CD11c (566504), anti-I-A/I-E (BioLegend, 107608), anti-CD80 (560016), anti-CD86 (561962), Brilliant Stain Buffer (563794), and FOXP3/Transcription Factor Staining Buffer Set (Invitrogen, 00-5523-00). Cells per 100 mg tumor were counted by Beckman Vi-Cell Auto. Flow cytometry was performed with Beckman CytoFLEX LX.
3
Immunophenotyping of Glioblastoma Tumor Samples
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Neoplastic tissues were collected from GBM patients and syngeneic intracranial GBM models, fixed with formalin, and embedded in paraffin. Frozen sections tumours were from obtained syngeneic intracranial GBM models. Paraffin-embedded sections and frozen sections were used for immunofluorescence staining. Primary antibodies, including anti-PD-L1 (Abcam, #224,030), anti-EMP3 (Invitrogen, PA5-97705), anti-CD86 (Abcam #239,075), anti-CD206 (Abcam, #125,028), anti-CD8α (Invitrogen, A700-044), anti-CD4 (Invitrogen, MA5-32166), and anti-Ki67 (CST, #12,075), were diluted (1:100) in PBS with 1 % BSA. After overnight incubation at 4 °C, the samples were washed three times with PBS and incubated with FITC-labelled anti-IgG (Alexa Fluor 488 or 594, Thermo Fisher) for 2 h at room temperature. DNA was stained with DAPI (Sigma-Aldrich, USA), and samples were visualized with a fluorescence microscope (Olympus, Japan). The proportion of stained cells/field was subjected to statistical analysis. All images were processed and analysed using ImageJ.
4
Multiparametric Flow Cytometry and Microscopy of Immune Cell Subsets
5
Immunohistochemistry of CD8+ T cells
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After blocking with 10% FBS (in PBS), the tumor slices were incubated with anti-CD8α (eBioscience) primary antibody for 1 h at room temperature and then visualized with a dye-conjugated secondary antibody under a Leica TCS-NT confocal microscope (Wetzler, Heidelberg, Germany).
6
Comprehensive Immune Cell Profiling by Flow Cytometry
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The following antibodies were used for flow cytometry analysis: anti-IFNγ (XMG1.2, 1:50), anti-CD45.1 (A20, 1:100), anti-CD45.2 (104, 1:100), anti-IL1β-APC (NJTEN3, 1:50), anti-CD44 (IM7, 1:100), anti-CD62l (MEL-14, 1:100), anti-F4/80 (BM8, 1:100), anti-CCR7 (4B12, 1:100), anti-CD4 (RM4-5, 1:200), anti-CD8α (53-6.7, 1:200), anti-H-2Kb (AF6-88.5.5.3, 1:200), anti-Ly6G (RB6-8C5, 1:100), anti-MHC class II (I-A/I-E) (M5/114.15.2, 1:100) and anti-CD11c (N418, 1:100) were from eBioscience. Anti-CD103 (2E7, 1:100) was from Biolegend. Annexin V apoptosis Detection kit (556547) was from BD Pharmingen. For intracellular cytokine staining, Cytofix/Cytoperm plus kit (BD, cat. 55508) was used. For Foxp3 staining, Foxp3 staining buffer set (eBioscience, cat. 00-5523-00) was used. Multiple-colour flow cytometric analysis was performed using FACSAria (BD Biosciences; Franklin Lakes, NJ, USA). For FACS sorting, cells stained with FACS
antibodies were sorted on BD FACSAria device. FlowJo software was used for data acquiring and analysis.
antibodies were sorted on BD FACSAria device. FlowJo software was used for data acquiring and analysis.
7
Isolation and Characterization of Intraepithelial Lymphocytes
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SI IELs were isolated as previously described7 (link) and GFP+ γδ IELs were sorted to 98% purity using a BD FACSAria II. IELs were stained with viability dye (eFluor 450 or eFluor 780), anti-CD3 (2C11), anti-CD8α (53–6.7), anti-CD8β (H35–17.2), anti-CD4 (GK1.5), anti-TCRβ (H57-597), anti-TCRγδ (GL3) (eBioscience), anti-Vγ7 (clone GL1.7; Rebecca O’Brien, National Jewish Health, Denver, CO), and anti-Vγ1 (clone 2.11, BioLegend) or a Click-iT Plus EdU Alexa Fluor 647 Flow Cytometry Assay Kit (Invitrogen). Flow cytometry was performed on an LSR Fortessa (BD Biosciences) in the New Jersey Medical School Flow Cytometry and Immunology Core Laboratory. Data were analyzed by FlowJo (v.10.4.0; Tree Star).
8
Flow Cytometry and Western Blot Antibodies
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The following antibodies, from BioLegend (San Diego, CA, USA) and eBioscience (San Diego, CA, USA), were used to stain cells for flow cytometry analysis: anti-B220 (RA3–6B2), anti-BrdU (BU20A), anti-CD3 (17A2), anti-CD4 (RM4–5 and GK1.5), anti-CD8α (53-6.7), anti-CD11b (M1/70), anti-CD11c (N418), anti-CD16/CD32 (93), anti-CD19 (eBio1D3), anti-CD25 (PC61.5), anti-CD34 (RAM34), anti-CD44 (IM7), anti-CD45.2 (104), anti-CD48 (HM48-1), anti-CD71 (R17217) anti-CD150 (TC15-12F12.2), anti-c-Kit (2B8), anti-Gr-1 (RB6–8C5), anti-IL-7Rα (A7R34), anti-Ki-67 (SolA15), anti-NK1.1 (PK136), anti-Sca-1 (D7) and anti-Ter119 (TER-119).
The following antibodies were used for western blotting: anti-p38 (D13E1), anti-phospho-p38 (D3F9), anti-p44/42 (137F5), anti-phospho-p44/42 (D13.14.4E), anti-JNK1 (2C6), anti-phopho-JNK and anti-β-actin (AC-15). Antibodies used to immunoblot MAPK signaling were from Cell Signaling Technology (Danvers, MA, USA) and anti-β-actin antibody was from Sigma-Aldrich (St Louis, MO, USA).
The following antibodies were used for western blotting: anti-p38 (D13E1), anti-phospho-p38 (D3F9), anti-p44/42 (137F5), anti-phospho-p44/42 (D13.14.4E), anti-JNK1 (2C6), anti-phopho-JNK and anti-β-actin (AC-15). Antibodies used to immunoblot MAPK signaling were from Cell Signaling Technology (Danvers, MA, USA) and anti-β-actin antibody was from Sigma-Aldrich (St Louis, MO, USA).
9
BrdU Uptake in Immune Cells
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BrdU was delivered to mice through drinking water at the concentration of 1mg/ml + 1% glucose. Spleen and lymph nodes were harvested on day 6 post-immunization or infection. Following tetramer enrichment, cells were stained with cell surface antibodies [anti-CD3, anti-CD4, anti-CD19 (eBio (1D3), eBiosciences), anti-CD8α, anti-CD11c (eBiosciences), anti-F4/80 (BM8, Biolegend) and anti-CD5 (53–7.3, BD Pharmingen)] followed by intracellular anti-BrdU (BD Pharmingen) antibody according to the BrdU flow kit protocol (BD Biosciences).
10
Isolation and Characterization of Intraepithelial Lymphocytes
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