Clone d4w2z, by Cell Signaling Technology - Product details

1

Quantification of CD8+ and CD163+ Cells

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Tissues were fixed for 24 h in 10% formalin and stored in 70% ethanol until further processing. Five micrometer sections of formalin fixed, paraffin-embedded tissues were deparafinized with xylene, rehydrated, and subjected to antigen retrieval with heated antigen unmasking solution (1.0 mM EDTA, 0.05% Tween-20, pH 8.0). After 1 h in horse serum blocking buffer, primary antibodies were applied for 3 h at room temperature or overnight at 4 °C. Anti-mouse antibodies included CD8a (1:400, Clone D4W2Z, Cell Signaling Technology) and CD163 (1:300, Clone EPR19518, Abcam).
For IHC, the ABC reagent was used with DAB chromogen, followed by counterstaining with hematoxylin QS (all from Vector Labs). Images were acquired with a Zeiss Axio Imager Z1. The number of cells showing CD8⁺or CD163⁺ in each high-power field (HPF) (×200) was quantitated.

Oba T., Long M.D., Keler T., Marsh H.C., Minderman H., Abrams S.I., Liu S, & Ito F. (2020). Overcoming primary and acquired resistance to anti-PD-L1 therapy by induction and activation of tumor-residing cDC1s. Nature Communications, 11, 5415.

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2

Immunofluorescent Profiling of Lung FFPE

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Lung FFPE blocks were cut into three-µm-thick sections and placed on slides. Slide sections were deparaffinized and rehydrated by serial passage through xylene and graded ethanol changes. Slides were subjected to antigen retrieval in AR9 or AR6 buffer (cat. AR9001KT, AR6001KT, Akoya Biosciences) and microwaved at 100% power for 60-90 seconds, then 20% power for 15 minutes. Then, slides were blocked and stained with rabbit monoclonal CD4 (cat. 25229S, clone D7D2Z, 1:400, Cell Signaling), rabbit monoclonal CD8 (cat. 98941S, clone D4W2Z, 1:400, Cell Signaling), rabbit monoclonal CD19 (cat. ab245235, clone EPR23174-145, 1:400, Abcam), rabbit monoclonal F4/80
(cat. ab111101, clone SP115, 1:400, Abcam), and rabbit monoclonal FOX/P3 (cat. 700914, clone 5H10L18, 1:400, Invitrogen) for 30 min at room temperature. The immunofluorescent signal was visualized using the OPAL™ 7-color Manual IHC kit (cat. NEL811001KT, Akoya Biosciences) and counterstained with Spectral DAPI. Then, slides were mounted with ProLongTM diamond antifade (cat. P36961, Invitrogen, CA, USA). All slides were scanned at high-powered 20x magnification using the Vectra Polaris^® (Perkin Elmer, MA). Cell phenotyping was analyzed with inForm^® Sofware v2.6 (Perkin Elmer, MA).

Kumpunya S., Thim-uam A., Thumarat C., Leelahavanichkul A., Kalpongnukul N., Chantaravisoot N., Pisitkun T, & Pisitkun P. (2022). cGAS deficiency enhances inflammasome activation in macrophages and inflammatory pathology in pristane-induced lupus. Frontiers in Immunology, 13, 1010764.

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3

Immunohistochemical Analysis of Tumor Organoids

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Tumour organoids were gently centrifuged at 100 rcf from organoid culture medium and then embedded in 2% agar. Tumour organoids were fixed in 4% paraformaldehyde solution for overnight at room temperature. The samples were replaced with 70% ethanol and later embedded in paraffin blocks. Paraffin sections were cut at 5 μm thickness, attached on slides, heated at 60°C for 1 h and then overnight at 37°C. The slides were stained with hematoxylin and eosin (H&E). IHC staining follows the protocol described^{62 (link)}. Mouse-specific antibodies for CD8α (Cell Signaling Technology, clone D4W2Z, 1:400 dilution), cleaved caspase3 (Cell Signaling Technology, clone D175, 1:400 dilution) and Ki67 (Cell Signaling Technology, clone D3B5, 1:400 dilution) were used for IHC staining. The DAB peroxidase substrate kit (Vector laboratories Inc.) was used to visualize the antibodies. For IF analysis, the sample slides were blocked 3% BSA for 40 min after hydration. The slides were stained with primary antibodies including EpCAM (Abcam, 1:400 dilution), FITC-conjugated anti-α-SMA (Genetex, 1:400 dilution) and AF594-conjugated anti-CD31 (Biolegend) for 1 h. For EpCAM staining, the slide was further stained with secondary antibody Alexa Fluor 647-onjugated anti-rabbit IgG (Biolegend, 1:200) for 40 min. The fluorescence images were captured under a Leica DM4B microscope.

Zhou Z., Van der Jeught K., Fang Y., Yu T., Li Y., Ao Z., Liu S., Zhang L., Yang Y., Eyvani H., Cox M.L., Wang X., He X., Ji G., Schneider B.P., Guo F., Wan J., Zhang X, & Lu X. (2021). An organoid-based screen for epigenetic inhibitors that stimulate antigen presentation and potentiate T-cell-mediated cytotoxicity. Nature biomedical engineering, 5(11), 1320-1335.

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Immunohistochemical Analysis of Tumor Organoids

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Tumour organoids were gently centrifuged at 100 rcf from organoid culture medium and then embedded in 2% agar. Tumour organoids were fixed in 4% paraformaldehyde solution for overnight at room temperature. The samples were replaced with 70% ethanol and later embedded in paraffin blocks. Paraffin sections were cut at 5 μm thickness, attached on slides, heated at 60°C for 1 h and then overnight at 37°C. The slides were stained with hematoxylin and eosin (H&E). IHC staining follows the protocol described^{62 (link)}. Mouse-specific antibodies for CD8α (Cell Signaling Technology, clone D4W2Z, 1:400 dilution), cleaved caspase3 (Cell Signaling Technology, clone D175, 1:400 dilution) and Ki67 (Cell Signaling Technology, clone D3B5, 1:400 dilution) were used for IHC staining. The DAB peroxidase substrate kit (Vector laboratories Inc.) was used to visualize the antibodies. For IF analysis, the sample slides were blocked 3% BSA for 40 min after hydration. The slides were stained with primary antibodies including EpCAM (Abcam, 1:400 dilution), FITC-conjugated anti-α-SMA (Genetex, 1:400 dilution) and AF594-conjugated anti-CD31 (Biolegend) for 1 h. For EpCAM staining, the slide was further stained with secondary antibody Alexa Fluor 647-onjugated anti-rabbit IgG (Biolegend, 1:200) for 40 min. The fluorescence images were captured under a Leica DM4B microscope.

Zhou Z., Van der Jeught K., Fang Y., Yu T., Li Y., Ao Z., Liu S., Zhang L., Yang Y., Eyvani H., Cox M.L., Wang X., He X., Ji G., Schneider B.P., Guo F., Wan J., Zhang X, & Lu X. (2021). An organoid-based screen for epigenetic inhibitors that stimulate antigen presentation and potentiate T-cell-mediated cytotoxicity. Nature biomedical engineering, 5(11), 1320-1335.

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5

Quantifying CD8+ Cells in Brain Tissue

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The frequency of Immunohistochemistry (IHC) staining was performed as described^{29 (link)}. To identify CD8⁺ cells in brain, anti-CD8α (1:400, Clone D4W2Z, Cell Signaling Technology) was used. Images were obtained with a Zeiss Axio Imager Z1. The number of cells showing CD8 positivity in each individual high-power field (HPF) (× 200) was quantified across five fields or more of nonsequential cryosections 9 mm in thickness.

Yokoi T., Oba T., Kajihara R., Abrams S.I, & Ito F. (2021). Local, multimodal intralesional therapy renders distant brain metastases susceptible to PD-L1 blockade in a preclinical model of triple-negative breast cancer. Scientific Reports, 11, 21992.

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Clone d4w2z

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5 protocols using clone d4w2z

Quantification of CD8+ and CD163+ Cells

Immunofluorescent Profiling of Lung FFPE

Immunohistochemical Analysis of Tumor Organoids

Immunohistochemical Analysis of Tumor Organoids

Quantifying CD8+ Cells in Brain Tissue

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