Tissue microarrays were constructed as described previously.26 (link) Immunohistochemical (IHC) staining was performed using mouse anti-human CD3 (Abcam, clone SP7), mouse anti-human CD8 (Dako, clone C8/144B), rabbit anti-human TIGIT (Abcam, clone BLR047F), and mouse anti-human CD56 (Dako, clone 123C3). TIL and IHC expression scores were determined by a blinded pathologist (MAD). TIL scores were calculated on H&E-stained slides as follows: 3 (>20 TIL/high-power field (hpf)), 2 (11–20 TIL/hpf), 1 (1–10 TIL/hpf), 0 (<1 TIL/hpf). IHC expression for CD3, CD8, CD56, and TIGIT was calculated as follows to derive an H-score: [1x (% 1+cells)+2x(% 2+cells)+3x(% 3+cells)] where 1+, 2+, and 3+ represent weak, moderate, and strong stain intensity, respectively. Final TIL and IHC expression scores were averaged when more than one consecutive section was present for scoring. PD-L1 staining of tumor samples was performed using a CLIA-certified laboratory.
Clone c8 144b
Manufactured by Abcam
Sourced in United States, Japan
Clone C8/144B is a mouse monoclonal antibody that recognizes human CD8 alpha. CD8 is a cell surface glycoprotein found on the surface of cytotoxic T cells and a subset of natural killer cells. This antibody can be used for the identification and enumeration of CD8+ T cells in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Clone sp7, by Agilent Technologies (1 mentions)
Clone 123c3, by Agilent Technologies (1 mentions)
Axiostar plus microscope, by Zeiss (1 mentions)
Ultrasensitive streptavidin peroxidase kit, by Maixin Group (1 mentions)
Bond max, by Leica (1 mentions)
Nanozoomer 2.0 ht scanner, by Hamamatsu Photonics (1 mentions)
View2, by Hamamatsu Photonics (1 mentions)
4 protocols using clone c8 144b
1
Immunohistochemical Profiling of Tumor-Infiltrating Lymphocytes
2
Immunohistochemical Analysis of Tumor Immune Infiltrates
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IHC analysis was performed on 4 µm thick slices of FFPE tumor specimens as previously described [15 (link)]. 7 patients had sufficient paired pre-treatment and post-treatment tumor tissue for transcriptomic analysis. Primary antibodies were used at—1:100 for CD8 (Agilent, clone C8/144B, Santa Clara, CA, USA), 1:200 for CD20 (Abcam, clone EP459Y, Cambridge, UK), and 1:100 for CD3 (Agilent, Clone F7.2.38, Santa Clara, CA, USA). A matched isotype was also applied on a replicate slide as a negative control. Corresponding secondary antibody were Envision+ System HRP anti-mouse IgG (Agilent, Santa Clara, CA, USA) for CD3, CD8 and anti-rabbit IgG (Agilent, Santa Clara, CA, USA) for CD20. For antigen retrieval, slides were heated for 20 min in Tris-EDTA-Tween buffer pH 9 for all antibodies. Scoring for CD8+ T cells was performed according to the Ovarian Tumor Tissue Analysis Consortium protocol [17 (link)] using a Zeiss Axiostar Plus microscope and Zen imaging software. A 4-point ordinal score was used based on CD8+ T cell counts per high powered field: negative (none), low (1–2), moderate (3–19), and high (≥20). The maximum score was used for each slide.
3
Immunohistochemical Analysis of Immune Cell Markers
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Tissues were harvested and fixed in 10% buffered formalin and embedded in paraffin for H&E staining. Serial sections (2.5 μm) were prepared for IHC examination of human CD4 (ABclonal Technology, clone 1H1R9, 1:500), CD8 (DAKO, clone C8/144B, 1:100), and CD31 (Abcam, ab28364, polyclonal). Immunoreactivity was detected with UltraSensitive TM Streptavidin‐Peroxidase Kit (Mai Xin, KIT‐9710) according to the manufacturer's protocol. The numbers of positively stained cells were assessed using the IHC profiler of Image J (NIH) and all the slides were observed and photographed with laser scanning confocal microscopy (LSM 880) using a 10× objective.
4
Quantifying CD69+ CD8+ T Cells in Liver
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IHC was performed using a Bond-Max automated immunohistochemical staining machine (Leica Microsystems, Milton Keynes, UK). Evaluation of CD69+ CD8+ T cells was performed using double fluorescent immunohistochemistry. Anti-human CD8 monoclonal Ab (Nichirei, Japan, clone C8/144B) and anti-human CD69 monoclonal antibody (Abcam, clone EPR21814) were used as primary Abs. The slides were scanned to obtain multi-colored whole-slide images using a NanoZoomer 2.0HT scanner (Hamamatsu Photonics K.K., Shizuoka, Japan) with a ×40 objective lens, according to the manufacturer’s instructions. The slides were further stained with H&E and rescanned to obtain merged images of H&E images on top of the pre-scanned multi-colored images51 (link). To quantify CD69+ CD8+ T cells, all portal areas were checked for merged CD69+ CD8+ T cells using NDP.view2 software (Hamamatsu Photonics K.K), and the highest number in 1-mm2 areas was determined as the maximum cell count. Detailed information for the antibodies used in this study is summarized in Supplementary Table 2 .
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