Cd68 clone pg m1

Manufactured by Agilent Technologies

Sourced in Denmark, United States, Germany

CD68 (clone PG-M1) is a laboratory reagent used in immunohistochemistry and flow cytometry applications. It is a mouse monoclonal antibody that recognizes the CD68 antigen, a glycoprotein expressed on the surface of macrophages and monocytes. This product can be used to identify and quantify macrophage populations in research samples.

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PG-M1 (51 citations) Anti-CD68 (clone PG-M1, (14 citations) Clone PG-M1 (13 citations) Anti-CD68 antibody (clone PG-M1, (5 citations) Anti-Human CD68 clone PG-M1 (5 citations) Monoclonal mouse anti-human CD68 clone PG-M1 (3 citations) Mouse anti-human CD68 (clone PG-M1) (3 citations) Mouse anti-CD68 (clone PG-M1, (2 citations)

36 protocols using cd68 clone pg m1

Quantitative Lymph Node Profiling

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For each patient, the whole available tissue specimen/block was cut and subsequent slides were stained in the Department of Pathology at Kiel University for CD3 (clone SP7, Waltham, MA, USA), CD20 (clone L26, Dako, Glostrup, Denmark), CD30 (clone BerH2, maintained at the Department of Pathology, Kiel, Germany) and CD68 (clone PG-M1, Dako, Glostrup, Denmark), using a Leica-Bond-Max stainer (Leica Microsystems, Wetzlar, Germany). The slides were scanned (Hamamatsu Nanozoomer, Hamamatsu Photonics, Ammersee, Germany) and the resulting images were processed by TissueStudio 64, according to the manufacturer’s recommendations (Definiens AG, Munich, Germany). The area ranged between 4-455 mm^{2 (link)} (mean: 133.81 mm^{2 (link)}, standard deviation [SD]: 80.84 mm^{2 (link)}) (Online Supplementary Figure S1). Since we included the entire lymph node in the analysis, any heterogeneity of cell distribution did not influence our data. Cutting artifacts, and overstained or unstained areas were manually excluded from the analysis. Adjusting the threshold based on several representative locations on the sample also ensured that the setting for analysis for each sample had been selected to cover the specific staining of the lymph node. See the Online Supplementary Methods for a detailed description and statistics.

Jachimowicz R.D., Pieper L., Reinke S., Gontarewicz A., Plütschow A., Haverkamp H., Frauenfeld L., Fend F., Overkamp M., Jochims F., Thorns C., Leo Hansmann M., Möller P., Rosenwald A., Stein H., Reinhardt H.C., Borchmann P., von Tresckow B., Engert A, & Klapper W. (2020). Whole-slide image analysis of the tumor microenvironment identifies low B-cell content as a predictor of adverse outcome in patients with advanced-stage classical Hodgkin lymphoma treated with BEACOPP. Haematologica, 106(6), 1684-1692.

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Multi-Modal Profiling of SARS-CoV-2 Infection

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In order to study different cells infected with SARS-CoV-2 virus RNAscope ISH was combined with conventional IHC staining with Alkaline Phosphatase Red using Bond Polymer Refine Red Detection Kit (DS9390). Directly after the ISH procedure the staining continued with IHC without further pretreatments. The antibody incubation time was doubled to 30 min from standard 15 minutes. Following antibodies were used: CD34 (clone QBEnd/10 form Dako # M7165, 1:25), CD68 (clone PG-M1 from Dako #M0876, 1:50), CK18 (clone DC-10 from Dako #M7010, 1:25), PDL1 (clone BSR90 from NordicBiosite # BSH-4003, 1:200), Granzyme B (clone 11F1 from Novocastra #NCL-L-GRAN-B, 1:25).

Szekely L., Bozoky B., Bendek M., Ostad M., Lavignasse P., Haag L., Wu J., Jing X., Gupta S., Saccon E., Sönnerborg A., Cao Y., Björnstedt M, & Szakos A. (2021). Pulmonary stromal expansion and intra-alveolar coagulation are primary causes of COVID-19 death. Heliyon, 7(5), e07134.

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Macrophage and PD-L1 Profiling in TMA

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TMA blocks were sliced into 4 μm‐thick sections, deparaffinized, and stained for CD68 (clone PG‐M1; Dako) as a pan macrophage marker and CD163 (clone 10D6; Leica Biosystems) as an M2 macrophage marker using a DAKO Autostainer Universal Staining System (Dako). In addition, TMAs were stained for PD‐L1 using PD‐L1 IHC 22C3 pharmDx assays (Agilent Technologies, Inc.) on an Autostainer Link 48 using an automated staining protocol.

Yanagawa N., Shikanai S., Sugai M., Koike Y., Asai Y., Tanji T., Sugimoto R., Osakabe M., Uesugi N., Saito H., Maemondo M, & Sugai T. (2023). Prognostic and predictive value of CD163 expression and the CD163/CD68 expression ratio for response to adjuvant chemotherapy in patients with surgically resected lung squamous cell carcinoma. Thoracic Cancer, 14(20), 1911-1920.

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Immunohistochemical Profiling of Skin Biopsies

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Four micrometer sections, cut from a 4-mm punch biopsy that were fixed in 10% buffered formalin and embedded in paraffin, were stained with hematoxylin and eosin (H&E) or labeled immunohistochemically with antibodies against CD4 (clone EP204, Epitomics, Cambridge, MA), CD8 (clone 4B11, Leica Biosystems Inc., Buffalo Grove, IL), CD56 (clone 56C04 Thermo-Scientific, Waltham, MA), CD68 (clone PG-M1, Dako, Carpinteria, CA) and CD138 (clone B-A38 Cell Marque, Rocklin, CA) using an automated immunohistochemistry staining platform (Bond Max, Leica-Microsystems, Buffalo Grove, IL). IHC detection of Tp was performed manually as previously described (10 (link)) using a rabbit polyclonal anti-Tp Ab (Biocare, Concord, CA, USA). Skin specimens from healthy volunteers of the same socioeconomic background and conditions were not available for analysis. Tissue known to contain Tp were used for the positive control of Tp IHCs. Additionally, secondary alone controls were used to assess any non-specific Ab binding for each Ab used.

Hawley K.L., Cruz A.R., Benjamin S.J., La Vake C.J., Cervantes J.L., LeDoyt M., Ramirez L.G., Mandich D., Fiel-Gan M., Caimano M.J., Radolf J.D, & Salazar J.C. (2017). IFNγ Enhances CD64-Potentiated Phagocytosis of Treponema pallidum Opsonized with Human Syphilitic Serum by Human Macrophages. Frontiers in Immunology, 8, 1227.

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Immunohistochemical Profiling of Ovarian Carcinoma

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Briefly, for immunohistochemistry, 4 μm sections were cut from the formalin-fixed paraffin embedded (FFPE) blocks of the cancer patient collected at the time of cytoreductive surgery and 79 additional high grade serous ovarian carcinoma from a recently described tissue microarray ovarian cancer cohort (15 (link)), and stained with the following antibodies according to the manufacturers’ instructions. CD8 (clone 144B, ready to use, DAKO, Carpinteria, CA), CD4 (clone 1F6, 1:40 dilution, Vector, Burlingame CA), CD3 (clone 2GV6, Ventana, Tucson, AZ), CD56 (clone 1B6, 1:200 dilution, Vector, Burlingame CA), CD68 (clone PG-M1, ready to use, DAKO, Carpinteria, CA), CD20 (clone L26, 1:200 dilution, DAKO, Carpinteria, CA), TIA-1 (clone TIA1, ready to use, Biocare, Concord, CA), CK7 (clone OVTL, Dako, Carpinteria, CA) and PD-L1 (clone E1L3N, 1:200 Cell Signaling, Danvers, MA). For antigen retrieval, the sections were pre-treated at low pH for PD-L1 and CD8, CD4, CD20, CD56 and CD68. PD-L1 antibody and membranous immunoreactivity was assessed semi-quantitatively in tumor cells as follows: <1% staining was considered negative, staining in 1–50% of tumor cells was scored as focal, and >50% staining was scored as diffusely positive.

Bellone S., Buza N., Choi J., Zammataro L., Gay L., Elvin J., Rimm D.L., Liu Y., Ratner E.S., Schwartz P.E, & Santin A.D. (2018). Exceptional response to pembrolizumab in a metastatic, chemotherapy/radiation-resistant ovarian cancer patient harboring a PD-L1-genetic rearrangement. Clinical cancer research : an official journal of the American Association for Cancer Research, 24(14), 3282-3291.

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Immunohistochemical Analysis of Carotid Arteries

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Paraffin-embedded carotid arteries were deparaffinized in xylene, rehydrated in graded ethanol, and subjected to immunostaining. Immunohistochemistry was performed on serial sections, as described previously [9 (link)]. The primary antibodies used were macrophage marker CD68 clone PG-M1 (Dako, Glostrup, Denmark), smooth muscle actin (SMC) clone 1A4 (Dako), ferritin, TfR (Dako), and thrombin receptor (protease-activated receptor 1, Sigma, Saint Louis, USA). The immunoreactions were visualized using the EnVision+/horseradish peroxidase (Dako) method and ChemMate EnVision Detection Kit (Dako). Control sections without primary antibodies or with non-immune IgG were run for each protocol, resulting in consistently negative results. The slides were counterstained with hematoxylin.
All histological sections were examined under a light microscope, and the images were digitalized with the Image Grabber program (Toronto, ON, Canada). The microscope was set on the same parameters used to scan all samples. The randomly digitalized images were analyzed with Adobe Photoshop (v5.5) as described previously [9 (link)]. The individual responsible for the analysis was blinded to patient information.

Li W., Osman E., Forssell C, & Yuan X.M. (2022). Protease-Activated Receptor 1 in Human Carotid Atheroma Is Significantly Related to Iron Metabolism, Plaque Vulnerability, and the Patient’s Age. International Journal of Molecular Sciences, 23(12), 6363.

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Immunostaining of Humanized Mice Tissues

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Tissues harvested from humanized and non-humanized NSG mice were fixed
with 4% paraformaldehyde for 24 hours and paraffin-embedded for immunostaining
with the indicated antibodies. Human CD45 (clone 2B11+PD2/26) and CD68 (clone
PGM1) antibodies were purchased from Dako (USA) and human CD3 and mouse Gr-1
antibodies were provided by MD Anderson histology core facility. Histology
slides were scanned by Aperio imaging system (Leica Biosystems, USA) and
analyzed using ImageScope software (Leica Biosystems, USA).

Meraz I.M., Majidi M., Meng F., Shao R., Ha M.J., Neri S., Fang B., Lin S.H., Tinkey P.T., Shpall E.J., Morris J, & Roth J.A. (2019). An improved patient-derived xenograft humanized mouse model for evaluation of lung cancer immune responses. Cancer immunology research, 7(8), 1267-1279.

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Immunohistochemical Profiling of Ovarian Cancer

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ICC was performed using antibodies against the following proteins: WT1 (clone 6F-H2; Dako, Santa Clara, CA, USA), PAX8 (clone MRQ-50; CellMarque, Darmstadt, Germany), calretinin (colne DAK-Calret1; Dako), luminal cytokeratin 19 (CK19, clone A53-B2.26; Selmark), CD44 (clone DF1485; Dako), CD68 (clone PG-M1; Dako), EpCAM (clone BerEP4; Dako), FN1 (Dako), and POSTN (Abcam). Reactions were performed on cytospined cell preparations of five established ovarian cancer lines and early and late passages of OVPA8 cultures.
Detection systems were used as follows: ImmPRESS™ REAGENT Anti-Rabbit Ig (Vector, Burlingame, CA, USA) for FN1 and POSTN, and EnVisionTM FLEX+, Mouse, High pH, and (Link) System (Dako) for other proteins. FN1 and POSTN were additionally detected using diaminobenzidine (DAB Peroxidase Substrate Kit, Vector) substrate.
For microscopic evaluation (Axiophot Microscope; Zeiss, Oberkochen, Germany), the preparations were counterstained with hematoxylin and mounted with Dako Ultramount Aqueous Permanent Mounting Medium (Dako). Pictures were acquired using a Pannoramic 250 Flash II scanner (3DHISTECH). The intensity of staining was evaluated as follows: − = negative; + = weak (few to 30% of cells); ++ = moderate (31–60%); +++ = strong (61–100%).

Tudrej P., Olbryt M., Zembala-Nożyńska E., Kujawa K.A., Cortez A.J., Fiszer-Kierzkowska A., Pigłowski W., Nikiel B., Głowala-Kosińska M., Bartkowska-Chrobok A., Smagur A., Fidyk W, & Lisowska K.M. (2018). Establishment and Characterization of the Novel High-Grade Serous Ovarian Cancer Cell Line OVPA8. International Journal of Molecular Sciences, 19(7), 2080.

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Liver Biopsy Immunohistochemical Analysis

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Liver biopsies were obtained for pathological examination. Standard morphological evaluation was based on hematoxylin and eosin (H&E) sections of formalin fixed specimens. To characterize the inflammatory infiltrates, immunohistochemical markers for macrophages (CD68, clone PG-M1, Dako, Glostrup, Denmark; CD163, clone 10D6, Thermo Scientific, Fremont, CA, USA), T lymphocytes (CD8, clone C8/144B, Dako, Glostrup, Denmark) and erythroid cells (Glycophorin, clone JC159, Dako, Glostrup, Denmark) were applied. Four-micron thick tissue sections were deparaffinised with xylene and rehydrated with graded alcohols. After washing in distilled water, sections were immersed in 3% hydrogen peroxide to block endogenous peroxidase.

Prencipe G., Bracaglia C., Caiello I., Pascarella A., Francalanci P., Pardeo M., Meneghel A., Martini G., Rossi M.N., Insalaco A., Marucci G., Nobili V., Spada M., Zulian F, & De Benedetti F. (2019). The interferon-gamma pathway is selectively up-regulated in the liver of patients with secondary hemophagocytic lymphohistiocytosis. PLoS ONE, 14(12), e0226043.

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Immunohistochemical Evaluation of Claudin-2

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Single- or double-staining procedures were applied for the TMA material with the antibodies to claudin-2 (Thermo Fisher Scientific Cat# 32-5600), CD68 (clone PG-M1; Dako, Inc., Denmark), and pan-cytokeratin antibody (clone AE1/AE3; Dako, Inc., Denmark). The abundance of the claudin-2 in tumor cells was evaluated as an integrated score, considering intensity of the expression and percentage of positive cells, using a four-graded scale (negative (0), weak (1), moderate (2), or strong (3)) and then dichotomized for survival analysis. Claudin-2-positive macrophage score was based on the quantity of marker-positive macrophages, irrespective of expression intensity. Claudin-2-positive CAF score was based on the abundance of dot-like expression (quantity of dots per area) irrespective of expression intensity. Evaluation of the ICH was performed by two pathologists: IH for SPCRC cohort (blinded to clinical and outcome data of SPCRC cohort) and AM for NORDIC-VII cohort (blinded to clinical and outcome data of NORDIC-VII).

Mezheyeuski A., Strell C., Hrynchyk I., Guren T.K., Dragomir A., Doroshenko T., Pashkova O., Gorgun J., Ruksha K., Pfeiffer P., Kure E.H., Sorbye H., Edler D., Martling A., Glimelius B., Östman A, & Portyanko A. (2017). Treatment-related survival associations of claudin-2 expression in fibroblasts of colorectal cancer. Virchows Archiv, 472(3), 395-405.

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