Anti cd20

Manufactured by Agilent Technologies

Sourced in Belgium, Japan, United States

Anti-CD20 is a monoclonal antibody used in laboratory applications. It binds to the CD20 antigen expressed on the surface of B cells, which is important for various immunological processes. The product is intended for research use only and its specific applications should be determined by the end-user.

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Anti-CD20 (clone L26; (9 citations) Mouse anti-human CD20 (5 citations) Anti-human CD20 (clone L26; (3 citations) Anti‐CD20 (L‐26; (3 citations) Mouse anti‐CD20 antibody (2 citations) CD20 mouse anti-human antibody (2 citations) Mouse anti-CD20 (clone L26, (2 citations) Anti-TM (1 citations)

20 protocols using anti cd20

Immunohistochemical Characterization of iehAM

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Explanted iehAM were fixed in 4% formaldehyde, embedded in paraffin, and stained with hematoxylin and eosin according to standard protocols. Additional sections were deparaffinized and pretreated with Ultra CC1 (Ventana Medical Systems, Tucson, AZ, USA) for antigen retrieval for anti-CD45, anti-CD20, anti-CD68, and anti-CD3 antibody staining and with target retrieval solution pH 9 (Dako, Heverlee, Belgium) for anti-pan-cytokeratin and anti-GFAP antibody staining. After endogenous peroxidase blocking, sections were incubated with 1:25 anti-CD45 (Dako, Heverlee, Belgium, clone 2B11 + PD7/26), 1:200 anti-CD20 (Dako, clone L-26), 1:1000 anti-CD68 (Merck, Darmstadt, Germany), 1:100 anti-pan-cytokeratin (Dako, clone 6F2), 1:200 anit-GFAP (Dako, clone AE1/AE3), and 1:20 anti-CD3 (Monosan, Uden, Netherlands, clone PS-1) antibodies. After incubation with peroxidase-labelled secondary antibodies, sections were visualized with a chromogen DAB (3,3′-diaminobenzidine) solution.

Hillenmayer A., Wertheimer C.M., Gerhard M.J., Priglinger S.G., Ohlmann A, & Wolf A. (2023). Epiretinal Amniotic Membrane in Complicated Retinal Detachment: a Clinical and In Vitro Safety Assessment. Ophthalmology and Therapy, 12(3), 1635-1648.

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Quantifying Immune Cell Profiles in Breast Cancer

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Quantification of immune cells by pathologists was performed as previously described (79 (link)). Staining of formalin-fixed paraffin-embedded tissue sections (4 μm thick) was performed with a BenchMark XT IHC/ISH automated slide stainer (Ventana Medical Systems Inc.). The antibodies used for immunohistochemical staining were anti-CD45 (Dako Denmark A/S), anti-CD3 (Dako Denmark A/S), and anti-CD20 (Dako Denmark A/S). They were revealed with the ultraView Universal DAB Detection Kit (Ventana Medical Systems Inc.). All staining reagents used were manufactured by Roche (F. Hoffmann–La Roche Ltd.). Images were analyzed with VisiomorphDP software (Visiopharm) to quantify the CD45⁺, CD3⁺, and CD20⁺ areas within the invasive tumor area defined for each digital image. The total positively stained area was scored as a percentage of the defined region, and the mean percentage of the scores obtained by two or three different pathologists was calculated for each sample. TET1 expression data for the IHC samples were taken from the Affymetrix data set GSE20711. The BLBC samples from this cohort were then separated into two groups (TET1-low and TET1-high), the cutoff being chosen to optimize the significance for CD45⁺ quantification between the two groups. The same split was then used for CD3⁺ and CD20⁺ quantification.

Collignon E., Canale A., Al Wardi C., Bizet M., Calonne E., Dedeurwaerder S., Garaud S., Naveaux C., Barham W., Wilson A., Bouchat S., Hubert P., Van Lint C., Yull F., Sotiriou C., Willard-Gallo K., Noel A, & Fuks F. (2018). Immunity drives TET1 regulation in cancer through NF-κB. Science Advances, 4(6), eaap7309.

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Immunohistochemical Analysis of Angptl2 and TGF-β1 in Tissues

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LF tissue samples were fixed in 4% paraformaldehyde (PFA), embedded in paraffin, and sectioned. After the sections were pre-treated with Target Retrieval Solution, pH 9 (Tris/EDTA buffer, pH 9; Dako Japan, Co., Ltd., Tokyo, Japan), endogenous peroxidases were blocked using periodic acid (Nichirei, Tokyo, Japan). We used the following antibodies as primary antibodies: anti-human Angptl2 antibody [19] (link), anti-vimentin, anti-CD3, anti-CD15, anti-CD20, anti-CD68 (Dako Japan), anti-S100A4 (Abcam, Cambridge, UK), and anti-human p-Smad3 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). After treatment using EnVision + System-HRP-labeled Polymer (Dako Japan), the labeling was visualized using a Histofine 3,3′-diaminobenzidine (DAB) kit (Nichirei). For double immunofluorescent staining, anti-vimentin (Dako Japan) and anti-Angptl2 or anti-TGF-β1 (Abcam) were used as the primary antibodies, and Alexa Fluor 488-labeled anti-rabbit IgG and Alexa Fluor 594-labeled anti-mouse IgG (Life Technologies) were used as the secondary antibodies. Nuclei were counterstained with 4′, 6′-diamidino-2-phenylindole (DAPI).

Nakamura T., Okada T., Endo M., Kadomatsu T., Taniwaki T., Sei A., Odagiri H., Masuda T., Fujimoto T., Nakamura T., Oike Y, & Mizuta H. (2014). Angiopoietin-Like Protein 2 Induced by Mechanical Stress Accelerates Degeneration and Hypertrophy of the Ligamentum Flavum in Lumbar Spinal Canal Stenosis. PLoS ONE, 9(1), e85542.

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Immunohistochemistry Staining of Immune Cells

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The air-dried cytospin slides were fixed in 95% alcohol for 30 min, and immunohistochemistry was performed on automated Ventana Benchmark (Ventana System, Tucson, AZ, USA) or Leica Bond Max (Leica Microsystems, Buffalo Grove, IL, USA) using the Bond polymer refine detection HRP (Leica Biosystems, DS9800) method. The following antibodies were used: prediluted anti-CD3 (clone: LN10; Leica), anti-CD20 (dilution 1:1000, clone: L26; DAKO), prediluted anti-CD19 (clone BT51E; Leica), prediluted anti-CD4 (clone: 4B12; Leica) and CD8 (dilution 1:40, clone: C8/144B; DAKO).

Gelarden I., Nguyen J., Gao J., Chen Q., Morales-Nebreda L., Wunderink R., Li L., Chmiel J.S., Hrisinko M., Marszalek L., Momnani S., Patel P., Sumugod R., Chao Q., Jennings L.J., Zembower T.R., Ji P, & Chen Y.H. (2021). Comprehensive evaluation of bronchoalveolar lavage from patients with severe COVID-19 and correlation with clinical outcomes. Human Pathology, 113, 92-103.

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Quantifying Brain Immune Cell Populations

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Human brain sections were immunostained using standard techniques using an anti-CD20 (1:500; Dako, Cirpinteria, CA), anti-CD3 (1:500; Dako), anti-CD4 (1:500; Ventana Medical Systems, Tucson, AZ), anti-CD68 (1:500; Dako), and anti-CD8 (1:500; Ventana Medical Systems, Tucson, AZ) antibodies. Total CD20+, CD3+, CD4+, and CD8+ cells present in each slide (one section per patient, N = 7 patients with liquefactive necrosis strokes) were counted using a Motic BA410 light microscope coupled with a MoticamPro 282A camera (Motic Images Plus 2.0 software) and a 10× objective, and divided by the area of tissue sample present on the slide to obtain the number of positivity stained cells per cm².

Nguyen T.V., Frye J.B., Zbesko J.C., Stepanovic K., Hayes M., Urzua A., Serrano G., Beach T.G, & Doyle K.P. (2016). Multiplex immunoassay characterization and species comparison of inflammation in acute and non-acute ischemic infarcts in human and mouse brain tissue. Acta Neuropathologica Communications, 4(1), 100.

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Immunohistochemical Analysis of CD20 and FcRL4

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Staining was performed on 5 µm frozen acetone-fixed sections. Endogenous peroxidase activity was blocked with Bloxall Solution (Vector) and 2% casein was used to block non-specific staining. Sections were washed with phosphate buffered saline (PBS) between incubation steps. Sections were incubated with anti-CD20 (Dako), mouse anti-FcRL4 (Biolegend), or isotype-matched irrelevant controls (Jackson ImmunoResearch) for 1 h, rabbit anti-mouse Ig (Dako) for 10 min, and anti-rabbit Ig peroxidase (Vector Laboratories) for 30 min. Staining was developed using a diaminobenzidine substrate (Vector Laboratories). Slides were counterstained with haematoxylin and mounted with dibutyl phthalate xylene (DPX) (Sigma Aldrich).

Yeo L., Lom H., Juarez M., Snow M., Buckley C.D., Filer A., Raza K, & Scheel-Toellner D. (2014). Expression of FcRL4 defines a pro-inflammatory, RANKL-producing B cell subset in rheumatoid arthritis. Annals of the Rheumatic Diseases, 74(5), 928-935.

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Immunohistochemistry for Lymphoma Diagnosis

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Ten patients were subjected to conventional H&E preparation and immunohistochemical staining. Microscopy was performed by two pathology doctors (WJ and JNC) according to the 2008 WHO classification of lymphoma neoplasms, and then two senior doctors (QL and CKS) rechecked the microscopy and made the final diagnosis. Antibodies CK, LCA, CD3, CD45RO, CD20, CD79a, CD5, Cyclin D1, CD10, MUM1, Bcl2, Bcl6, CD30, and Ki-67 were obtained from Abcam USA and DAKO Denmark. The processes were as follows: immunohistochemical staining for 4-μm tissues were performed with anti-CK (1:100), anti-LCA (1:400), anti-CD3 (1:100), anti-CD45RO (1:500), anti-CD20 (1:250), anti-CD79a (1:100), anti-CD5 (1:100), anti-Cyclin D1 (RTU), anti-CD10 (1:50), anti-MUM1 (1:50), anti-Bcl2 (1:100), anti-Bcl6 (1:100), anti-CD30 (1:40), and anti-Ki-67 (1:100) and were detected using the DAKO En-Vision System (Dako Diagnostics, Zug, Switzerland) according to the manufacturer’s instructions.

Chen Z., Liang Q., Wang J., Huang Q.X., Chen J.N., Weng Z.J., Shao C.K., Gao X, & Pang J. (2018). Collapsin response mediator protein 4 promotor methylation level as a potential predictor for diagnosing primary malignant lymphoma of the prostate. Cancer Cell International, 18, 3.

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Automated Immunohistochemistry and EBER

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Staining and in situ hybridization were performed on an automated stainer (Bond Max; Leica Biosystems). The presence of EBV was demonstrated by in situ hybridization for the small RNA–encoding regions 1 and 2 (EBER). Antibodies used and dilution were anti-CD3 (polyclonal anti-CD3, 1/200; DAKO), anti-CD20 (polyclonal anti-CD3, 1/200; DAKO), and anti-CD8 (clone C8/144B; 1/200; DAKO).

Rodriguez R., Fournier B., Cordeiro D.J., Winter S., Izawa K., Martin E., Boutboul D., Lenoir C., Fraitag S., Kracker S., Watts T.H., Picard C., Bruneau J., Callebaut I., Fischer A., Neven B, & Latour S. (2019). Concomitant PIK3CD and TNFRSF9 deficiencies cause chronic active Epstein-Barr virus infection of T cells. The Journal of Experimental Medicine, 216(12), 2800-2818.

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Histological and Immunohistochemical Analysis of Skin Lesions

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Formalin-fixed tissue samples were stained with H&E for histological description. Masson-Fontana (MF) staining was performed for characterization of the pigmentation pattern. Immunohistochemical procedures were performed on 20 representative SL derived in equal parts from the hand and the face. Tissue samples were ﬁxed in 4% formaldehyde overnight and embedded in paraffin. Paraffin-embedded blocks were sectioned at a 6-μm thickness and deparaffinized in xylene, hydrated with ethanol, and rinsed in distilled water. Citrate buffer incubation (40 min, 10 mM citric acid, 0.05% Tween 20, pH 6) in a steamer was used for epitope retrieval. Afterwards, sections were cooled for 20 min and rinsed in phosphate-buffered saline. The following primary antibodies were used: anti-melan A (1: 60; Leica Microsystems), anti-tyrosinase (1: 60; Novocastra), anti-MITF (clone D5, 1: 60; Dako) p53 (1: 60; Dako), and anti-CD20 (1: 60; Dako). Notch1 immunofluorescence staining (Cell Signaling Technology) was performed on 12 of those lesions (5 lesions derived from the face and 7 lesions from the hand), according to a previously described method [14 (link)]. All tissue samples were microscopically observed in ×40 magnification (Olympus BX51TF).

Barysch M.J., Braun R.P., Kolm I., Ahlgrimm-Siesz V., Hofmann-Wellenhof R., Duval C., Warrick E., Bernerd F., Nouveau S, & Dummer R. (2019). Keratinocytic Malfunction as a Trigger for the Development of Solar Lentigines. Dermatopathology, 6(1), 1-11.

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Quantitative Analysis of Lymph Node Follicular Structures

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Hematoxylin and eosin stained lymph node sections were analyzed at low-power (12.5X) on a DM4000B microscope (Leica Biosystems), to capture the nodes entire two-dimensional area with a Spot RT/SE Slider camera (Spot Imaging). Follicular structures were traced and scored for circularity in silico with ImageJ software (NIH freeware) using the formula:
circularity=4π(area/perimeter²)
Lymph node follicular composition was determined by dividing the combined 2-dimensional areas of all follicular structures by the total lymph node area excluding adipose rich or acellular fibrous tissues. All pathology images were reviewed by one or more hematopathologists. Immunohistochemical staining was performed with anti-CD21 (1:100, Dako), anti-CD3 (pre-diluted, Ventana), anti-CD20 (1:200, Dako) and anti-BCL6 (1:50, Dako) antibodies.

Romberg N., Le Coz C., Glauzy S., Schickel J.N., Trofa M., Nolan B.E., Paessler M., Xu M.L., Lambert M., Lakhani S.A., Khokha M.K., Jyonouchi S., Heimall J., Takach P., Maglione P.J., Catanzaro J., Hsu F.I., Sullivan K.E., Cunningham-Rundles C, & Meffre E. (2018). CVID patients with autoimmune cytopenias exhibit hyperplastic yet inefficient germinal center responses. The Journal of allergy and clinical immunology, 143(1), 258-265.

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