Ab4059

Manufactured by Abcam

Sourced in United Kingdom, China, United States

Ab4059 is a rabbit polyclonal antibody. It is designed for use as a control in immunological assays.

Automatically generated - may contain errors

Lab products found in correlation

Ab183685, by Abcam (5 mentions) Ab209775, by Abcam (4 mentions) Pd l1, by Cell Signaling Technology (3 mentions) Ab205921, by Abcam (3 mentions) Ab52632, by Abcam (2 mentions) Pano multiplex ihc kit, by Panovue (2 mentions) Panck, by Cell Signaling Technology (2 mentions) Ab16669, by Abcam (2 mentions) Ab111101, by Abcam (2 mentions) Ab33786, by Abcam (2 mentions) Aperio cs2 slide scanner, by Leica (1 mentions) Ab181551, by Abcam (1 mentions) Vp k451, by Vector Laboratories (1 mentions) Ab91279, by Abcam (1 mentions) Apotome 2, by Zeiss (1 mentions) Ab52488, by Abcam (1 mentions) Ab93684, by Abcam (1 mentions) Axio observer light microscope, by Zeiss (1 mentions) Ab150077, by Abcam (1 mentions) Bx 63 upright fluorescent microscope, by Olympus (1 mentions)

29 protocols using ab4059

Immunohistochemical Analysis of Autoimmune Bullous Diseases

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Paraffin-embedded skin samples from patients affected by bullous pemphigoid (N = 3), dermatitis herpetiformis (N = 3), and inflammatory EBA (N = 3) were sectioned (5 μm) for immunohistochemical analysis of GzmB (Cat # ab4059, ABCAM) and collagen VII (Cat # ab93350, ABCAM, Toronto, ON) using 3,3′-Diaminobenzidine for visualization, as well as α6 (Cat # ab181551, ABCAM) and β4 (Cat # ab182120, ABCAM) integrins, and collagen XVII (NC16a-3^{70 (link)}) visualized through Novared®. Healthy skin obtained from patients undergoing elective abdominoplasty was used as a control. In order to observe cellular infiltrate and tissue architecture, H&E staining was also performed using established methods. Following image acquisition, these H&E stained sections were de-stained by serial incubation in xylene (2 min), 100% EtOH (2 min), 95% EtOH (2 min), water (10 min), and acidic alcohol solution (10 min). In order to detect GzmB-producing cells, de-stained H&E sections were probed for GzmB (Cat # ab4059, ABCAM). All slides were scanned using a Aperio CS2 slide scanner (Leica, Concord, ON).

Russo V., Klein T., Lim D.J., Solis N., Machado Y., Hiroyasu S., Nabai L., Shen Y., Zeglinski M.R., Zhao H., Oram C.P., Lennox P.A., Van Laeken N., Carr N.J., Crawford R.I., Franzke C.W., Overall C.M, & Granville D.J. (2018). Granzyme B is elevated in autoimmune blistering diseases and cleaves key anchoring proteins of the dermal-epidermal junction. Scientific Reports, 8, 9690.

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Immunohistochemical Analysis of Tissue Samples

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Four-micrometer sections of formalin-fixed, paraffin-embedded tissues were prepared for conventional IHC and stained for 45 minutes with the rabbit anti-human granzyme B polyclonal Ab (Abcam, ab4059) 1:100, mouse anti-human PD-L1 mAb 10.4 μg/mL (Clone 405.9A11, (Boston, MA)) or rabbit anti-human Ki67 polyclonal Ab 1:2000 (Vector, VP-K451), followed by secondary HRP conjugated anti-rabbit or anti-mouse Ab. The anti-PD-L1 mAb used for IHC [21 (link)] recognizes a different domain than the anti-PD-L1 mAb secreted by the lentivirus. The slides were developed using 3,3′-diaminobenzidine (DAB) and counterstained with hematoxylin. The quantification of the IHC images was performed using the IHC Profiler Plugin of ImageJ Software [47 (link)].

Suarez E.R., Chang D.K., Sun J., Sui J., Freeman G.J., Signoretti S., Zhu Q, & Marasco W.A. (2016). Chimeric antigen receptor T cells secreting anti-PD-L1 antibodies more effectively regress renal cell carcinoma in a humanized mouse model. Oncotarget, 7(23), 34341-34355.

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Multiplex IHC for Immune Cell Profiling

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Two 4-µm sections from TMA blocks were subjected to mfIHC using the PANO Multiplex IHC kit (0004100100, Panovue, Beijing, China) to examine specific cell markers including CD11c (ab52632, Abcam), CD45RO (55618, Cell Signaling), CD68 (ZM0060, ZSGB-Bio), panCK (4545, Cell Signaling), and PD-L1 (13684, Cell Signaling) in panel A, and CD4 (BX50023, biolynx), CD8A (70306, Cell Signaling), CD56 (3576, Cell Signaling), FoxP3 (320202, Biolegend), and granzyme B (ab4059, Abcam) in panel B. Different primary antibodies were sequentially applied, followed by horseradish peroxidase-conjugated secondary antibody incubation and TSA. The slides were microwave heat-treated after each TSA operation. Nuclei were stained with 4ʹ-6ʹ-diamidino-2-phenylindole (DAPI, D9524, Sigma-Aldrich) after all the human antigens had been labeled.

Pan C., Wang Y., Liu Q., Hu Y., Fu J., Xie X., Zhang S., Xi M, & Wen J. (2021). Phenotypic profiling and prognostic significance of immune infiltrates in esophageal squamous cell carcinoma. Oncoimmunology, 10(1), 1883890.

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Immunostaining of Murine Melanoma Tumors

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Deparaffinization and antigen retrieval were performed in murine melanoma subcutaneous tumors using Antigen Retrieval Buffer (Abcam ab52488 and ab93684) and stained as previously described.^{20 (link)} The following antibodies were used for immune staining as described previously:^{5 (link)} anti-CD3 (ab16669), anti-CD4 (ab183685), anti-CD8 (ab22378), anti-granzyme B (ab4059), anti-F4/80 (ab6640), anti-iNOS (ab3523), anti-Arg.1 (ab91279), anti-LDHA (ab52488), and Goat Anti-Rabbit Alexa 488 (ab150077), all antibodies were purchased from Abcam. Images were obtained by using an Axio Observer Light Microscope with the Apotome.2 (Zeiss). Metastatic melanoma lesions were gated by generating a region of interest, and threshold merged fluorescence limited to ROI and calculated using the ImageJ software.

de Azevedo R.A., Shoshan E., Whang S., Markel G., Jaiswal A.R., Liu A., Curran M.A., Travassos L.R, & Bar-Eli M. (2020). MIF inhibition as a strategy for overcoming resistance to immune checkpoint blockade therapy in melanoma. Oncoimmunology, 9(1), 1846915.

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Histological and Immunohistochemical Characterization of Organoids

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Organoids were fixed for histology on days 1 and 10 of culture in 4% paraformaldehyde for 4 hours. Organoids were processed, paraffin embedded, and sectioned at 5-μm intervals for staining. Organoid sections were stained on glass microscope slides with hematoxylin and eosin (H&E).
Additional staining was performed with immunohistochemistry (IHC) to characterize programmed death ligand-1 (PD-L1), cluster of differentiation 8 (CD-8), cytokeratin 20 (CK-20), and granzyme B biomarker expression. Unstained slides underwent antigen retrieval in a pH 6 citrate buffer solution prior to blocking with Dako Protein Block for 30 minutes. Fluorescent IHC was performed by applying primary antibodies PD-L1 (ab205921, abcam, rabbit), CD-8 (ab4055, abcam, rabbit), CK-20 (MA5–13263, Invitrogen, mouse), and granzyme B (ab4059, abcam, rabbit), and cleaved caspase 3 (9661S, Cell Signaling Technologies, rabbit) to slides in ratios of 1:500, 1:200, 1:200, 1:100, 1:400 in Dako Antibody Diluent, respectively. After incubation for 1 hour, appropriate species reactive secondary Alexa Fluor 488 or Alexa Fluor 594 antibodies (Biotium, Fremont, CA) were applied to samples for 1 hour at a 1:1000 dilution. Sections were then incubated with DAPI for 5 minutes prior to finalization with coverslipping. An Olympus BX-63 upright fluorescent microscope (Olympus, Tokyo, Japan) was used to image the sections.

Forsythe S.D., Erali R.A., Sasikumar S., Laney P., Shelkey E., D’Agostino R J.r., Miller L.D., Shen P., Levine E.A., Soker S, & Votanopoulos K.I. (2021). Organoid Platform in Preclinical Investigation of Personalized Immunotherapy Efficacy in Appendiceal Cancer: Feasibility Study. Clinical cancer research : an official journal of the American Association for Cancer Research, 27(18), 5141-5150.

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High-Grade Serous Ovarian Cancer Profiling

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Ovarian cancer tissue samples were obtained with informed consent from 41 treatment naïve HG‐SOC patients at the Tongji Hospital of HUST, and the baseline information of these patients is applied in Table S1. The specimens were extracted during surgery, and tissues were formalin‐fixed and paraffin‐embedded. Standard IHC was performed using antibodies against granzyme B (Abcam, ab4059), CD68 (Abcam, ab125212), F4/80 (Servicebio, GB113373), PD‐L1 (Abcam, ab238697), and IL‐1β (Cell Signaling Technology, 12242S) using an Avidin‐Biotin Complex Vectastain Kit (Zsgb‐Bio, SP‐9000) according to the manufacturer's protocols. Tissue sections were scanned after immunostaining, and protein expressions were measured using Image J software. The diagnosis of high‐grade serous ovarian cancer (HG‐SOC) was confirmed by two independent pathologists.

Xu C., Xia Y., Zhang B., Drokow E.K., Li H., Xu S., Wang Z., Wang S., Jin P., Fang T., Xiong X., Huang P., Jin N., Tan J., Zhong Q., Chen Y., Zhang Q., Fang Y., Ye F, & Gao Q. (2023). Macrophages facilitate tumor cell PD‐L1 expression via an IL‐1β‐centered loop to attenuate immune checkpoint blockade. MedComm, 4(2), e242.

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Immunohistochemical Analysis of Breast Tumors

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Human breast tumour tissue specimens were obtained following the guidelines approved by the Institutional Review Board at MD Anderson Cancer Center, and written informed consent was obtained from patients in all cases at the time of enrolment. IHC staining was performed as described previously39 (link)42 (link). Briefly, tissue specimens were incubated with antibodies against PD-L1 (Cell Signaling Technology, #13684, 1:30 dilution), p-EGFR (Y1068; #3777, Cell Signaling Technology, 1:200 dilution), p-GSK3β (S9; #9323, Cell Signaling Technology, 1:200 dilution) or Granzyme B (ab4059, Abcam, 1:100 dilution) and a biotin-conjugated secondary antibody and then incubated with an avidin–biotin–peroxidase complex. Visualization was performed using amino-ethylcarbazole chromogen. For statistical analysis, Fisher's exact test and Pearson χ²-test were used and a P value less than 0.05 is considered statistically significant. The intensity of staining was ranked into four groups: high (+++), medium (++), low (+) and negative (−) according to histological scores.

Li C.W., Lim S.O., Xia W., Lee H.H., Chan L.C., Kuo C.W., Khoo K.H., Chang S.S., Cha J.H., Kim T., Hsu J.L., Wu Y., Hsu J.M., Yamaguchi H., Ding Q., Wang Y., Yao J., Lee C.C., Wu H.J., Sahin A.A., Allison J.P., Yu D., Hortobagyi G.N, & Hung M.C. (2016). Glycosylation and stabilization of programmed death ligand-1 suppresses T-cell activity. Nature Communications, 7, 12632.

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Antibodies for Western Blot and IHC Analysis

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The primary antibodies used in western blot analysis included PD‐L1 anti‐human (#13684, CST), PD‐L1 anti‐mouse (17952‐1‐AP, Proteintech), ALDH2 (15310‐1‐AP, Proteintech), Myc tag (#2278, CST), Flag tag (20543‐1‐AP, Proteintech), HA tag (51064‐2‐AP, Proteintech), SPOP (16750‐1‐AP, Proteintech), and GAPDH (60004‐1‐Ig, Proteintech). Antibodies used in IHC assay were anti‐mouse CD3 (ab16669, Abcam), anti‐mouse CD8 (ab209775, Abcam), anti‐mouse granzyme B (ab4059, Abcam), anti‐human CD3 (Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China), and anti‐human CD8 (Zhongshan Golden Bridge Biotechnology Co., Ltd.). The secondary antibodies used in immunofluorescence assay were AF488‐anti‐rabbit (#8878, CST) and AF594‐anti‐mouse (Biotech). MG‐132 (M8699) and chloroquine (C6628) were purchased from Sigma. CHX (HY12320) was purchased from MedChem Express.

Zhang H., Xia Y., Wang F., Luo M., Yang K., Liang S., An S., Wu S., Yang C., Chen D., Xu M., Cai M., To K.K, & Fu L. (2021). Aldehyde Dehydrogenase 2 Mediates Alcohol‐Induced Colorectal Cancer Immune Escape through Stabilizing PD‐L1 Expression. Advanced Science, 8(10), 2003404.

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Immunohistochemical Analysis of NK Cells

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Harvested tumor tissues were embedded in optimal cutting temperature (OCT) solution (Fisher Scientific). Serial 4-μm thick sections were made for IHC staining. The tumor tissue sections were dried at RT overnight and fixed with 4% paraformaldehyde for 10 min at room temperature (RT). The tumor tissue sections were permeabilized with 0.1% Triton X-100/1× PBS for 10 min, followed by a wash with 3 × 0.1% BSA/1× PBS for granzyme B detection. The tumor tissue sections were blocked with 5% BSA in PBS for 30 min and 3% H₂O₂ (3 drops) for 10 min at RT. Anti-mouse CD49b (NK cell marker) (Invitrogen, 14-5971-85) and anti-mouse granzyme B (Abcam, ab4059) were applied as primary antibodies at a concentration of 5 μg/ml. A biotinylated anti-rat antibody (Vector lab, BA-4001) was used at 5 μg/ml for secondary antibody detection with the ABC Peroxidase Detection System according to the manufacturer’s instructions. Three tumor tissue sections (n = 3) were stained from each mouse group. Tumor sections were scanned under ×200 (magnification = 20 × 10) to identify all positive stained cells using an Olympus DP72 with microscopy equipped with an imaging processing software. Positive tissue staining was quantitated using Image J software.

Fan X., Yuan Z., Zhao Y., Xiong W., Hsiao H.C., Pare R., Ding J., Almosa A., Sun K., Zhang S., Jordan R.E., Lee C.S., An Z, & Zhang N. (2022). Impairment of IgG Fc functions promotes tumor progression and suppresses NK cell antitumor actions. Communications Biology, 5, 960.

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IRIN Silicasome Tumor Therapy Evaluation

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Tumor‐bearing mice received IV injection to deliver an IRIN dose of 40 mg kg⁻¹ (free IRIN or IRIN silicasome) per injection every 3 days for a total of 3 administrations. Control animals received saline only. Animals were sacrificed 72 h after the last injection. To confirm the impact on tumor growth, ex vivo bioluminescence imaging was performed to assess image intensity at the primary and metastatic tumor sites. Primary tumors were fixed in 10% formalin, followed by paraffin embedding and sectioning to provide 4 µm slices for IHC analysis in the UCLA Translational Pathology Core Laboratory (TPCL). The slides were scanned and images were assessed by using Aperio ImageScope software (Leica). Primary antibodies to CD8 (#14‐0808‐82) and FoxP3 (#13‐5773‐82) were purchased from ThermoFisher; CRT (ab2907), while antibodies to HMGB1 (ab18256), granzyme B (ab4059), perforin (ab16074) and IFN‐γ (ab9657) were purchased from Abcam. The antibody to LC‐3 (#0231‐100/LC3‐5F10) was purchased from Nanotools, while the antibody to PD‐L1 (#64988) was purchased from Cell Signaling Technology.

Liu X., Jiang J., Liao Y., Tang I., Zheng E., Qiu W., Lin M., Wang X., Ji Y., Mei K., Liu Q., Chang C.H., Wainberg Z.A., Nel A.E, & Meng H. (2021). Combination Chemo‐Immunotherapy for Pancreatic Cancer Using the Immunogenic Effects of an Irinotecan Silicasome Nanocarrier Plus Anti‐PD‐1. Advanced Science, 8(6), 2002147.

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