Gb13068

Manufactured by Wuhan Servicebio Technology

Sourced in China

GB13068 is a laboratory equipment product. It is designed for general laboratory use. The core function of this product is to assist with scientific research and experiments, but a detailed description cannot be provided while maintaining an unbiased and factual approach.

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GB13068-2 (2 citations)

5 protocols using gb13068

Immune Profiling of CAR-T/M-THP1 and hMSC Therapy

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Mice were sacrificed on day 10 after CAR-T/M-THP1 and hMSC injection, and tumor samples were fixed with formalin and embedded in paraffin. Tumor tissues were examined by immunohistochemistry staining as previously described (Jiang et al., 2018 (link)). Briefly, the sections were exposed to 3% H₂O₂ in methanol after deparaffinization and rehydration and then blocked with 1% BSA for 30 min at room temperature. After blocking, the sections were incubated with primary antibodies (all from Servicebio Technology Co., China) for IL-1β (GB11113), CD4⁺ (GB13064-1), CD8⁺ (GB13068), and FOXP3 (GB11093) overnight at 4°C, followed by incubation with peroxidase-conjugated secondary antibodies. IL-1β+ cells were quantified by measuring the number of stained cells.

Zhang R., Liu Q., Zhou S., He H., Zhao M, & Ma W. (2023). Mesenchymal stem cell suppresses the efficacy of CAR-T toward killing lymphoma cells by modulating the microenvironment through stanniocalcin-1. eLife, 12, e82934.

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Immunohistochemical Analysis of Tumor Samples

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To assess histopathological changes, tumor tissues were fixed with 4% paraformaldehyde and embedded in paraffin wax. Tissues were sliced into 4 μm-thick tumor sections and then stained with hematoxylin/eosin (H&E) for visualization of the tissue structure. For the IHC staining assay, the tissue sections were deparaffinated and incubated with 5% bovine serum albumin for 60 min. Then the tissue sections were incubated with anti-CD3 antibody (1:100, Servicebio, GB130144-M), anti-CD4 antibody (1:100, Servicebio, GB13064) or anti-CD8 antibody (1:100, Servicebio, GB13068) for 2 h, respectively. After incubation, 3% H₂O₂ was used to eliminate the activity of endogenous peroxidase and Goat anti-rabbit lgG was used as a secondary antibody. 3,3′-diaminobenzidinetetrahydrochloride (DAB-4HCl) was used to visualize the CD3, CD4 and CD8 expression. Images were acquired using a Nikon Eclipse 80i microscope (Nikon, Badhoevedorp, The Netherlands). Tissue evaluation was performed by two independent examiners and semi quantitated by image J Software.

He C., Zhou Y., Li Z., Farooq M.A., Ajmal I., Zhang H., Zhang L., Tao L., Yao J., Du B., Liu M, & Jiang W. (2020). Co-Expression of IL-7 Improves NKG2D-Based CAR T Cell Therapy on Prostate Cancer by Enhancing the Expansion and Inhibiting the Apoptosis and Exhaustion. Cancers, 12(7), 1969.

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Multiplex IHC with Fluorescent Staining

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TSA plus fluorescent multiple staining kit (#G1236-100T, Servicebio) was applied for multiplex IHC. The 5-mm formalin-fixed and parrffin-embedded slides were deparaffinized and rehydrated. All slides were subjected to epitope retrieval with EDTA (pH 8.0) for 8 min. After cooling (25℃), slides were washed with PBS (3*5 min), and endogenous peroxidase activity were blocked with H₂O₂ (3%) for 25 min. Then blocking buffer (5%BSA, Solarbio, #SW3015) was used for 30 min-protein blocking. All slides were incubated with antibody against CD8 (#GB13068, Servicebio, 1:500) at 4℃ overnight. After washes, sections were incubated with an HRP-conjugated secondary antibody for 50 min. Then slides were dyed with CY3-TSA for 10 min. This method was applied three more times using the antibodies as follows, GINS2 (Proteintech, #16247-1-AP, 1:1000, dye FITC), CD4 (Wisee Biotechnology, #YX32005-100, 1:1000, dye 647-TSA), and CD3 (Servicebio, #GB13440, 1:100, dye594). EDTA (pH 8.0) buffer was applied for the next round of epitope retrieval with a cooker (125℃, 15 min). Cell nucleus was labeled with DAPI (Servicebio, #G1012) and covered with Antifade Mounting Medium (Beyotime, #P0126). Secondary antibodies were used as follows: anti-rabbit (1:500, Servicebio, GB23303) and anti-mouse (1:500, Servicebio, GB23301).

Li Z., Song G., Guo D., Zhou Z., Qiu C., Xiao C., Wang X, & Wang Y. (2022). Identification of GINS2 prognostic potential and involvement in immune cell infiltration in hepatocellular carcinoma. Journal of Cancer, 13(2), 610-622.

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Immunohistochemical and Immunofluorescence Analysis of Tumor Samples

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Tissue samples were fixed, treated, and stained according to standard procedures. Simply, the tumor was excised from mice, fixed with 4% neutral buffer formaldehyde, and embedded in paraffin at the Institute of Biopathology (Servicebio, China). The primary antibodies used for immunohistochemistry staining were a rabbit anti-human CD3 monoclonal antibody (176171-AP, Proteintech) and a rabbit anti-human IFN-γ monoclonal antibody (ab9657, Abcam). Immunohistochemistry (IHC) section images were obtained using a microscope (Leica, DM6000B). The primary antibodies used for immunofluorescence staining were as follows: rabbit anti-human CD8 antibody (GB13068, Servicebio), rabbit anti-human IFN-γ antibody (GB11107-1, Servicebio), and mouse anti-human GZMB antibody (GB14092, Servicebio). Fluorescent secondary antibodies were as follows: goat anti-rabbit antibody labeled with horseradish peroxidase (HRP; GB23303, Servicebio), goat anti-mouse antibody labeled with HRP (GB23301, Servicebio), and goat anti-mouse antibody labeled with CY5 (GB27301, Servicebio). DAPI (G1012, Servicebio) was used for staining of the nucleus. Pathological section scanners (Pannoramic, 3Dhistech) were used to detect fluorescence signals. To quantify the immunofluorescence results, the average fluorescence intensity was analyzed using ImageJ software.

Xia B., Lin K., Wang X., Chen F., Zhou M., Li Y., Lin Y., Qiao Y., Li R., Zhang W., He X., Zou F., Li L., Lu L., Chen C., Li W., Zhang H, & Liu B. (2023). Nanobody-derived bispecific CAR-T cell therapy enhances the anti-tumor efficacy of T cell lymphoma treatment. Molecular Therapy Oncolytics, 30, 86-102.

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Multiplex Immunofluorescence Staining Protocol

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Paraffin-embedded samples were dewaxed, hydrated, heat treated for antigen repair, incubated with primary antibody, and added with corresponding fluorescent secondary antibody. The primary and secondary antibodies were washed off and incubated with the second primary antibody and the corresponding fluorescent secondary antibody. DAPI-counterstained (G1012, Servicebio, Wuhan, China) nuclei were incubated at room temperature for 10 min. Spontaneous fluorescence quenching reagent (G1221, Servicebio) was added and incubated for 5 min to quench the autofluorescence of tissues. After the slices were slightly dried, they were sealed with antifade mounting medium (G1401, Servicebio). The sections were observed under a fluorescence microscope, and the images were analyzed by CaseViewer (version 2.4.0).
The following antibodies were used to detect the corresponding proteins: CD14 (GB14023, dilution 1:100, Servicebio), CD68 (GB113150, dilution 1:200, Servicebio), CD3 (GB14176, dilution 1:100, Servicebio), and CD8 (GB13068, dilution 1:200, Servicebio). The used fluorescent secondary antibodies include CY3 goat anti-mouse antibody (GB21301, dilution 1:300, Servicebio) and 488 goat anti-rabbit antibody (gb25303, dilution 1:400, Servicebio).

Luo Y., Tao T., Tao R., Huang G, & Wu S. (2022). Single-Cell Transcriptome Comparison of Bladder Cancer Reveals Its Ecosystem. Frontiers in Oncology, 12, 818147.

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