Epr19518

Manufactured by Abcam

Sourced in Germany, United States, China

EPR19518 is a recombinant monoclonal antibody that specifically recognizes Erk1/2 (MAPK3/MAPK1). The antibody is designed for use in various immunoassay applications to detect and quantify Erk1/2 protein levels.

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Lab products found in correlation

7 protocols using epr19518

Multiplex Immunofluorescence Staining Protocol

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Each antibody was validated using conventional immunohistochemistry and monoplex immunofluorescence staining in conjunction with the corresponding opal fluorophore and the spectral DAPI counterstain. The antibodies were tested at three different dilutions, starting with manufacturer recommended dilution (MRD), MRD/2, and MRD/4 with 1/100 tyramide to select the optimal concentration of antibody capable of generating the best signal. The signal was then optimized through different tyramide titrations until generation of the best exposure time ranging from 5 to 25ms. Reproducibility was evaluated using: a positive control of each antibody with DAPI and a DAPI-alone slide. The negative controls included one unstained slide for auto-fluorescence compensation, a tyramide-alone slide treated with hydrogen peroxide for endogenous peroxidase masking, and finally a secondary antibody with tyramide slide. The following antibodies were used in the multiplex panel analysis: S100B Leica NCL-L-S100167 1/100 pH6 with Tyr480 1/200; pSTAT3 Cell Signaling Tyr705 D3A7 XP 1/200 pH 6 with Tyr520 1:150; CD68 Agilent PG-M1 1:50 pH 9 with Tyr570 1:150; CD3 Agilent clone F7.2.38 1:50 with pH 6 buffer for antigen retrieval with Tyr620 1:300; CD163 Abcam EPR19518 1:500 pH 9 with Tyr690 1:100; and CD11c Abcam EP1347Y 1:300 pH 9 with Tyr780 1:100.

Najem H., Marisetty A., Horbinski C., Long J., Huse J.T., Glitza Oliva I.C., Ferguson S.D., Kumthekar P.U., Wainwright D.A., Chen P., Lesniak M.S., Burks J.K, & Heimberger A.B. (2021). CD11c+CD163+ Cells and Signal Transducer and Activator of Transcription 3 (STAT3) Expression Are Common in Melanoma Leptomeningeal Disease. Frontiers in Immunology, 12, 745893.

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Immunofluorescence Staining of Meningioma

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Immunofluorescence staining was performed as described previously.^{17 (link)} Briefly, 4 µm tissue sections were cut in series from formalin-fixed paraffin-embedded tissue blocks for each meningioma case. Standard H&E staining was performed for each tumor and histological features of each section were reviewed by a neuropathologist establishing the WHO grade (Neuropathology report). For immunofluorescence, sections were stained overnight with primary antibodies; mouse anti-CD68 antibodies (1:200, (KP1) conjugated to Alexa-488 Santa Cruz Biotechnology), mouse anti-CD80 (1:100, (R&D Systems), mouse anti-CD86 (1;100, D-6 Santa Cruz Biotechnology together with either rabbit anti-CD163 (1:200, EPR19518, Abcam), rabbit anti-CD206 (1:1000, ab64693, Abcam), or rabbit anti-iNOS (1:200, Abcam). Secondary goat anti-rabbit or anti-mouse Alexa-555 & -488 antibodies (Molecular probes) were applied to sections and tissue auto-fluorescence was blocked with 30-min incubation in 0.02% Sudan black in 70% EtOH. Sections were then coverslipped using hard-set mounting media containing DAPI (Vectashield).

Proctor D.T., Huang J., Lama S., Albakr A., Van Marle G, & Sutherland G.R. (2019). Tumor-associated macrophage infiltration in meningioma. Neuro-oncology Advances, 1(1), vdz018.

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Immunohistochemistry Protocol for FFPE Samples

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Sections were deparafinized in Xylene and Ethanol and rinsed in water. Heat‐induced epitope retrieval with Tris‐EDTA, pH = 9 was performed for 2.5 h in a steamer. Sections were washed in TBS, permeabilized with 0.2% Tween, washed, and blocked for 1 h in 5% BSA (Jackson Immunoresearch, Hamburg, Germany) in TBS. An endogenous avidin/biotin blocking kit (Abcam) was used if biotin‐streptavidin amplification was employed. The same primary antibodies were used for FFPE as for frozen sections, aside from the mouse anti‐perforin antibody (5B10, dilution, 1:30; Dianova, Hamburg, Germany) and the rabbit anti‐CD163 antibody (EPR19518, dilution 1:200; Abcam). Isotype control stainings were performed to ensure specificities.

Konjevic Sabolek M., Held K., Beltrán E., Niedl A.G., Meinl E., Hohlfeld R., Lassmann H, & Dornmair K. (2019). Communication of CD8+ T cells with mononuclear phagocytes in multiple sclerosis. Annals of Clinical and Translational Neurology, 6(7), 1151-1164.

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Multiplexed Immunohistochemistry for PD-L1 and Immune Cells

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Immunohistochemistry studies were performed on formalin-fixed and paraffin-embedded (FFPE) tissue sections collected by the study sponsors at the time of the trials. An in-house double IHC staining assay was developed using an extensively validated antibody against PD-L1 (405. 9A11 mouse monoclonal antibody, 1:100, 13 micrograms/ml, Dr. Freeman laboratory, Dana-Farber Cancer Institute, Boston, MA, USA and commercially available through Cell Signaling Technology (CST))(23 (link)-27 (link)) and a cocktail of antibodies recognizing immune cells consisting of anti-CD45 (1:500, D9M8I XP, rabbit monoclonal antibody, CST) with anti-CD163 (1:5000, EPR19518, rabbit monoclonal antibody, Abcam). Tumor sections were stained with Bond Rx Autostainer (Leica Biosystems, Buffalo Grove, IL) using the Bond Polymer Refine Detection Kit (DS9800; Leica Biosystems) and Bond Polymer Refine Red Detection Kit (DS9390, Leica Biosystems). Antigen retrieval was performed with Bond Epitope Retrieval Solution 2 (EDTA, pH = 9.0) for 30 minutes. All slides were counterstained with hematoxylin, dehydrated in graded ethanol and xylene, mounted, and cover slipped (Supplementary Figure 1).

Flaifel A., Xie W., Braun D.A., Ficial M., Bakouny Z., Nassar A.H., Jennings R.B., Escudier B., George D.J., Motzer R.J., Morris M.J., Powles T., Wang E., Huang Y., Freeman G.J., Choueiri T.K, & Signoretti S. (2019). PD-L1 expression and clinical outcomes to cabozantinib, everolimus and sunitinib in patients with metastatic renal cell carcinoma: analysis of the randomized clinical trials METEOR and CABOSUN. Clinical cancer research : an official journal of the American Association for Cancer Research, 25(20), 6080-6088.

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Antibody Panel for Cell Differentiation

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Antibodies used in this study are as follows: Recombinant Anti-CD163 antibody [EPR19518; abcam], recombinant Anti-CD68 antibody [EPR20545; abcam], Recombinant Anti-CD11b antibody [EP1345Y]—C-terminal (ab52478; abcam), CD61 (Integrin beta 3) Recombinant Rabbit Monoclonal Antibody (SJ19-09; Thermofisher scientific), CD41 mouse Monoclonal Antibody (CRC64; Thermofisher scientific), Rabbit Polyclonal Anti-Syncytin-1 antibody (Clinisciences), Anticorps mouse monoclonal RUNX2 (F-2; Santa Cruz), and Anti-Telomerase reverse transcriptase antibody (MA5-16033, Thermofisher).

Ali A.M., BenMohamed F., Decina A., Mukherjee S., Levi S., Garrido Castillo L.N., Bréchot D., Jurcic J., Raza A, & Paterlini Bréchot P. (2023). Circulating cancer giant cells with unique characteristics frequently found in patients with myelodysplastic syndromes (MDS). Medical Oncology (Northwood, London, England), 40(7), 204.

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Comprehensive Immunophenotyping of Tumor Specimens

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By reviewing the H&E slides, a representative tissue block that contained the tumor and adjacent normal tissues was chosen for IHC staining in each case. On the SWTSs from the blocks above, IHC staining for CD20 (OTI4B4, ZSGB-Bio, Ready-to-use), IgD (EPR6146, Abcam, 1:3,500), CD21 (EP3093, Abcam, 1:500), CD23 (EPR3617, Abcam, 1:400), CD8 (SP16, ZSGB-Bio, Ready-to-use), FOXP3 (EPR22102-37, Abcam, 1:250), PD-1 (EPR4877(2), Abcam, 1:500), EOMES (EPR21950-241, Abcam, 1:1,000), NCR1 (EPR22403-57, Abcam, 1:1,000), CD163 (EPR19518, Abcam, 1:500), and PD-L1 (E1L3N, Cell Signaling Technology, 1:200) was performed with the two-step polymer-based detection system (PV-8000, ZSGB-Bio). IHC slides passing the quality control were scanned into a computer with an Aperio Digital Pathology Slide Scanner (Aperio Technologies, Vista, CA, USA) at ×200 magnification for subsequent analyses.

Zou Y., Li D., Yu X., Zhou C., Zhu C, & Yuan Y. (2023). Correlation of Neuroendocrine Differentiation with a Distinctively Suppressive Immune Microenvironment in Gastric Cancer. Neuroendocrinology, 114(2), 192-206.

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Immunohistochemical Analysis of Colorectal Samples

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Colorectal samples were fixed in 4% paraformaldehyde. Tissues were embedded in paraffin and cut into 4-μm sections. The sections were submerged in xylene at 40ºC for 30 min for deparaffinization. Deparaffinized tissues were dehydrated in a gradient of alcohol solutions (100%, 95%, 80% and 75%) for 1 min in each. The sections were washed in distilled water for 1 min and then stained with haematoxylin and eosin (HE). After washing three times in PBS for 3 min, the deparaffinized slides were submerged in 120℃ distilled water containing citrate (1: 100) or EDTA (1: 50) for 2 min and incubated for 20 min at room temperature for antigen retrieval. Endogenous peroxidase activity was blocked with 1.0% H₂O₂ for 10 min. The sections were incubated with mouse anti-human CD68 (KPI, ab955, Abcam), rabbit anti-human HLA-DR (EPR3692, ab92511, Abcam) and rabbit anti-human CD163 (EPR19518, ab182422, Abcam) monoclonal antibodies at 4°C overnight, followed by incubation with a secondary antibody (Max Vision^TM HRP kit-5020, MXB, China). The HRP-conjugated secondary antibody was visualized by development with diaminobenzidine (DAB; DAB-0031/1031, MXB, China). All sections were counterstained with haematoxylin.

Wang Z., Du Z., Sheng H., Xu X., Wang W., Yang J., Sun J, & Yang J. (2021). Polarization of intestinal tumour-associated macrophages regulates the development of schistosomal colorectal cancer. Journal of Cancer, 12(4), 1033-1041.

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