PA5-16701

antibody from Invitrogen Antibodies

Targeting: MS4A1 B1, Bp35, CD20, MS4A2

Provider product page for PA5-16701

Western blot

Antibody data

Antibody Data
Antigen structure
References [67]
Comments [0]
Validations
Western blot [2]
Immunocytochemistry [1]
Immunohistochemistry [5]
Flow cytometry [2]
Other assay [20]

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Product number: PA5-16701 - Provider product page
Provider: Invitrogen Antibodies
Product name: CD20 Polyclonal Antibody
Antibody type: Polyclonal
Antigen: Synthetic peptide
Description: Upon delivery aliquot and store at -20ºC, avoiding freeze/thaw cycles. For IHC staining, antigen retrieval is not essential but may optimize staining. Recommended positive controls: BJAB WCL and tonsil.
Reactivity: Human, Mouse, Canine, Feline
Host: Rabbit
Isotype: IgG
Vial size: 500 µL
Concentration: 0.077 mg/mL
Storage: -20° C, Avoid Freeze/Thaw Cycles

MS4A1 protein structure - PA5-16701 shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of CD20 was performed by loading 25 µg of Daudi (lane 1), Ramos (lane 2), Raji (lane 3) and BAF-3 (lane 4) cell lysates onto an SDS polyacrylamide gel. Proteins were transferred to a PVDF membrane and blocked at 4ºC overnight. The membrane was probed with a CD20 polyclonal antibody (Product # PA5-16701) at a dilution of 1:10,000 (Daudi and Ramos) and 1:200 (Raji and BAF-3) overnight at 4°C, washed in TBST, and probed with an HRP-conjugated secondary antibody for 1 hr at room temperature in the dark. Chemiluminescent detection was performed using Pierce ECL Plus Western Blotting Substrate (Product # 32132). Results show a band at ~33 kDa.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-CD20 Polyclonal Antibody, (Product # PA5-16701) and 33 kDa band corresponding to CD20 was observed in the cell lines tested except in Jurkat, Hep G2 which are low expressing models. Mouse cell lines showed no reactivity. Membrane enriched cell extracts (40 µg lysate) of Raji (Lane 1), Ramos (Lane 2), Daudi (Lane 3), Jurkat (Lane 4), Hep G2 (Lane 5), SP2/0-Ag14 (Lane 6) and EL4 (Lane 7) were electrophoresed using Novex® NuPAGE® 4-12 % Bis-Tris gel (Product # NP0321BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blots were probed with the primary antibody (1:2000 dilution) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L), Superclonal™ Recombinant Secondary Antibody, HRP conjugate (Product # A27036, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Formalin-fixed, paraffin-embedded human tonsil stained with CD20 using peroxidase-conjugate and AEC. Note cell membrane staining of B lymphocytes

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemistry was performed on formalin-fixed, paraffin-embedded canine lymph node using an automated staining platform. Following deparaffinization, antigen retrieval was performed using CC1 retrieval solution for 30 minutes followed by 32 minute primary antibody incubation with rabbit anti-CD20 polyclonal antibody (Product # PA5-16701) at a dilution of 1:100. Detection was performed using an indirect biotin-streptavidin system and a DAB Detection kit. Data courtesy of the Innovators Program.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemistry was performed on formalin-fixed, paraffin-embedded canine B cell lymphoma using an automated staining platform. Following deparaffinization, antigen retrieval was performed using CC1 retrieval solution for 30 minutes followed by 32 minute primary antibody incubation with rabbit anti-CD20 polyclonal antibody (Product # PA5-16701) at a dilution of 1:100. Detection was performed using an indirect biotin-streptavidin system and a DAB Detection kit. Data courtesy of the Innovators Program.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemistry was performed on formalin-fixed, paraffin-embedded feline lymph node using an automated staining platform. Following deparaffinization, antigen retrieval was performed using CC1 retrieval solution for 30 minutes followed by 32 minute primary antibody incubation with rabbit anti-CD20 polyclonal antibody (Product # PA5-16701) at a dilution of 1:100. Detection was performed using an indirect biotin-streptavidin system and a DAB Detection kit. Data courtesy of the Innovators Program.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemistry was performed on formalin-fixed, paraffin-embedded feline B cell lymphoma using an automated staining platform. Following deparaffinization, antigen retrieval was performed using CC1 retrieval solution for 30 minutes followed by 32 minute primary antibody incubation with rabbit anti-CD20 polyclonal antibody (Product # PA5-16701) at a dilution of 1:100. Detection was performed using an indirect biotin-streptavidin system and a DAB Detection kit. Data courtesy of the Innovators Program.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 3 Representative CNS and PNS Histopathologic Findings in Rhesus Macaques Injected with Suboccipital AAV9.hIDUA (A-C) The majority of animals had minimal to mild neuronal cell body degeneration in the DRG characterized by neurodegeneration (arrow, A), satellitosis, and mononuclear cell infiltrates that surrounded and invaded neuronal cell bodies (arrows, B). A normal DRG from a vehicle control animal is represented for comparison (C). (D-F) All animals from test article-treated groups had an axonopathy in the dorsal white matter tracts of the spinal cord that was bilateral and characterized by dilated myelin sheaths with and without myelomacrophages (arrows), consistent with axonal degeneration. An unremarkable section from the vehicle control group is shown for comparison (F). H&E staining was used. (G) Individual cumulative scores defined as the sum of cervical, thoracic, and lumbar segments scores with 1 as minimal, 2 as mild, 3 as moderate, 4 as marked, and 5 as severe; no animal had a score higher than 3 in any segment analyzed. Error bars represent SDs. (H) Immunofluorescence staining of the DRG using anti-hIDUA antibodies (red), anti-CD20 or anti-CD3 antibodies (green), and DAPI (nuclear counterstaining). Mononuclear cell infiltrates were predominantly composed of CD3 + T cells (B) and CD20 + B cells (C). Scale bars, 200 mum (A-C) and 100 mum (D-F).

Supportive validation

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Experimental details: Figure 6 Lymphocytes traffic to peripheral lymphoid tissues after adoptive transfer. A Diagram displaying the transfer protocol. B Donor bulk PBMC were transferred from MCM7 into MCM6. MCM6 was euthanized 24 hours post transfer and its tissues were processed. Cells selectively trafficked to different peripheral lymphoid organs post-transfer. C Immunohistochemical staining of a lymph node section showing PKH67+ cells (green), CD20 antibody staining (red) and CD8 antibody staining (blue) collected with a confocal microscope at 200X.

Supportive validation

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Experimental details: FIGURE 6 Prolonged pharmacological MALT1 protease inhibition leads to reduced Tregs in dogs. (A) Outline of 4-week and 13-week GLP toxicity studies including pre-test, treatment and recovery phases. (B) Frequency of Foxp3 + CD25 + Tregs and (C) CD3 + T cells in blood and spleen in male and female beagle dogs determined by FACS at time of necropsy (day 86 or recovery day 55). Each dot represents an individual animal. Lines depict mean +- SD. Statistical difference was determined using one-way ANOVA with follow up for significance by multiple comparison tests with Sidak's correction,* p < 0.05. (D) Histological alterations in stomach of beagle dogs treated with MLT-943 for up to 13 weeks. All animals were females and belonged to either the vehicle or the 10-7 mg/kg/day group. Sections taken at necropsy day 86 and stained with H&E (left panel), anti-CD3 (middle panel), or anti-CD20 (right panel).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 1 Immunohistochemical analysis of lymphoid cells in the brains of -synuclein transgenic mice. a Representative bright field light microscopy images from the neocortex, hippocampus, striatum, and thalamus of non-tg and -syn tg mice immunostained with an antibody against CD3 (general T cell marker). b Computer-based image analysis showing significant increase of CD3 positive cell numbers in neocortex, hippocampus, and striatum of -syn tg mice. c Representative bright field light microscopy images from the neocortex, hippocampus, striatum, and thalamus of non-tg and -syn tg mice immunostained with an antibody against CD20 (general B cell marker). d Computer-based image analysis showing comparable numbers of CD20 positive cell in the brains of non-tg and -syn tg mice. e Representative bright field light microscopy images from the neocortex, hippocampus, striatum, and thalamus of non-tg and -syn tg mice immunostained with an antibody against CD4 (helper T cell marker). f Computer-based image analysis showing significant increase of CD4 positive cell numbers in neocortex, hippocampus and striatum of -syn tg mice. g Representative bright field light microscopy images from the neocortex, hippocampus, striatum, and thalamus of non-tg and -syn tg mice immunostained with an antibody against CD8 (cytotoxic T cell marker). h Computer-based image analysis showing significant increase of CD8 positive cell numbers in neocortex, hippocampus, and striatum of -syn tg mice. Scale bars = 40

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 4 Immunohistochemical analysis of lymphoid cells in the brains of DLB cases. a Representative bright field light microscopy images from the neocortex and hippocampus of control and DLB cases immunostained with an antibody against CD3 (general T cell marker). b Computer-based image analysis showing significant increase of CD3 positive cell numbers in neocortex and hippocampus in DLB. c Representative bright field light microscopy images from the neocortex and hippocampus of control and DLB immunostained with an antibody against CD20 (general B cell marker). d Computer-based image analysis showing very few or none CD20-positive cell in the brains. e Representative bright field light microscopy images from the neocortex and hippocampus of control and DLB cases, immunostained with an antibody against CD4 (helper T cell marker). f Computer-based image analysis showing significant increase of CD4-positive cell numbers in neocortex and hippocampus in DLB. g Representative bright field light microscopy images from the neocortex and hippocampus in control and DLB immunostained with an antibody against CD8 (cytotoxic T cell marker). h Computer-based image analysis showing few CD8-positive cells in human brains. i , j Linear regression analysis between CD3 and CD4, CD8, and CD20 in the neocortex and hippocampus. Scale bars = 40 mum (low magnification) and 10 mum (high magnification). Control and DLB ( n = 8 per group). Statistical significance determined by unpaired t test; * p

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 4 Immunohistochemical (IHC) stained histological slides of different types of primary antibody--( a ) CD3, ( b ) CD20, ( c ) IgG antibody. A/F--abutment-fixture junction; upper (blue) and lower (maroon) region of interest (ROI) selected for cell marker intensity analysis.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 2 ( A ) Microscopic lesion of tumor mass, uniform proliferation of lymphoblast and small to medium lymphocyte, HE, Scale bar = 50 um; ( B ) HE, Scale bar = 20 um; ( C ) IHC CD3, diffuse positive staining, Scale bar = 50 um; ( D ) IHC CD20, negative staining, Scale bar = 50 um.

Supportive validation

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Experimental details: Figure 3 ( A ) Microscopic lesion of cecal tonsils, loss of intestine structure, caused by neoplastic proliferation and massive infiltration of lamina propria, HE, Scale bar = 250 um; ( B ) Lymphoblasts with pleomorphic nuclei, and small to medium in size lymphocytes, HE Scale bar = 50 um; ( C ) IHC CD3, diffuse positive staining, Scale bar = 100 um; ( D ) IHC CD3 Scale bar= 50 um; ( E ) IHC CD20, negative staining, Scale bar = 1000 um; ( F ) IHC CD20, Scale bar = 50 um.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 6. The tc aptamer controlled CD20 expression. ( A ) Schematic representation of the suicide system. When treated with the therapeutic antibody rituximab (RTX), cell death was induced in cells expressing CD20. As a positive control, CD20 was stably integrated into HeLa HF1-3 cells (cell line HF1-3CD20). The aptamer-controlled exon 2 along with the neighboring introns from construct L2 was inserted into the CD20 coding region, which resulted in the cell line HF1-3C2. In the presence of tc, exon 2 was skipped, the coding sequence of CD20 was restored, and apoptosis was triggered. Without tc, exon 2 was retained, which destroyed the CD20 reading frame and resulted in cell survival. ( B, C ) Cell lines were incubated with and without 50 muM tc for 2 days. Afterwards, RT-PCR and a western blot were performed. Primers used for RT-PCR bind to the coding sequence of CD20, which spans the alternatively spliced exon 2. HSP60 was used as a loading control for western blot. ( D ) Viability assay. The cell lines were incubated with and without RTX and 50 muM tc or doxycycline (dox) for 3 days. Afterwards, cell viability was analyzed by an AlamarBlue (r) assay. Error bars represent the standard deviation from mean values. For each cell line, the value of untreated cells was set to 100. The values of untreated cells are shown in Supplementary Figure S6 .

Supportive validation

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Experimental details: Figure 1 Intra-species differences in the numbers of macrophages (based on Iba1 immunostaining), plasma cells (lambda chain), T lymphocytes (CD3) and B lymphocytes (CD20) in skin mange lesions from wild carnivores and ruminants. For each animal species, the same letters above different rectangular bars indicates significant differences between means. For wolf: (a, b, c) p < 0.001. For fox: (a, b, c, d, e) p < 0.05. For chamois: (a, b) p < 0.05. For red deer: (a, b, c) p < 0.001. A Tukey test for multiple comparisons was applied for statistical analysis.

Supportive validation

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Experimental details: Figure 2 Immunohistochemistry to detect microglia, T lymphocytes, and B lymphocytes in thalamus ( a - c ) and hippocampus ( d - f ). Microglia were identified based on labeling with anti-Iba1 antibodies. Abundant cells are visible within glial foci ( a ) and in the perivascular infiltrate ( d ). T lymphocytes were identified based on labeling with anti-CD3 antibodies. Moderate numbers are visible in the glial focus ( b ) and in the perivascular cuff ( e ). B lymphocytes were identified based on labeling with anti-CD20 antibodies. Few numbers are visible within the glial focus ( c ) and perivascular infiltrate ( f ). Erythrocytes were also immunolabelled (asterisk) but were not included in the counting.

Supportive validation

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Experimental details: Figure 2 Feline cutaneous non-epitheliotropic B-cell lymphoma. (a) A population of medium and large round elements has been highlighted, with abundant cytoplasm and a round-to-cleaved nucleus with prominent and irregular nucleolus (May-Grunwald-Giemsa [MGG], x 40). (b) Several binucleated (arrow) cells can be seen (MGG, x 60). (c) Medium and large round neoplastic cells infiltrate extensively the superficial and deep dermis (haematoxylin and eosin [H&E], x 20). (d) There was no evidence of epitheliotropism in the neoplastic cells (H&E, x 40). (e-g) Neoplastic cells show strong positivity to CD20 marker (immunohistochemistry [IHC], x 20, x 40). (f-h) Few cells show positivity to marker CD3 (IHC, x 20, x 40)

Supportive validation

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Experimental details: Fig. 2. Comparison of the rapid multiple immunofluorescence (RMIF) CD20/CD3 and RMIF CD79alpha/CD3 methods. A & B: Detection of CD20. C & D: Detection of CD79alpha. A & C: Fine needle biopsy samples from the lymph node of a dog with T-cell lymphoma. B & D: Sediment smear from the chylous effusion of a cat. In the RMIF CD20/CD3 samples, green signals for CD20 were observed in the margins of certain lymphocytes, and negative reactions were clearly observable in most lymphocytes. In the RIMF CD79alpha/CD3 samples, the weak, nonspecific green signals were detected in multiple lymphocytes. In all panels, a fluorescence filter adapted to Alexa 488 was used. Scale bars=10 um.

Supportive validation

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Experimental details: Figure 1 Design and effect of poly(A) track insertion on CD20 stability and expression (A) Scheme representative of poly(A) insertions and location of insertions for CD20 reporter constructs used in this study. Poly(A) insertions in the CD20 coding sequence were placed proximal to the ATG start codon. A schematic of the CD20 gene with the start codon (ATG), termination codon (STOP), and exons and insertion sites is also shown. (B) Western blot analysis of CD20 constructs expressed transiently in CHO cells. An equal amount of total protein was used for analysis. GFP-YFP and beta-actin were used as transfection and loading controls, respectively, with expression of both proteins detected using specific antibodies. CD20 was detected with a CD20-specfic antibody. Arrows indicate the position of full-length CD20, GFP-YFP, and beta-actin proteins. (C) mRNA levels of CD20 constructs were measured by qRT-PCR. CD20 expression was normalized to beta-actin. Relative mRNA levels for both constructs (12As and 18As) are presented as percentages of WT CD20. Error bars represent mean +- SD (n = 3). p values represent significance (*p

Supportive validation

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Experimental details: FIGURE 2 Sections of immunostaining with a polyclonal anti-CD20 + mab of canine mammary sections from animals affected by breast cancer. (A) CD20 + Lymphocyte infiltrating tumor sample (20X) in a G1 sample; (B) CD20 + Lymphocyte infiltrating tumor sample (20X) in a G2 sample; (C) CD20 + Lymphocyte infiltrating tumor sample (20X) in a G3 sample.

Supportive validation

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Experimental details: Figure 8 CD20 immunostained sections in the spleen showing A: control group shows CD20+ B cells appearing mainly in lymphatic follicles of white pulp (arrow). B: TAA-treated group showing increased CD20+ B cells in lymphatic follicles of white pulp (black arrow) and in red pulp (red arrow). C: rapamycin-treated group showing CD20+ B cells in lymphatic follicles of white pulp (black arrow) and few in the red pulp (red arrow). D: Filgrastim-treated group showing CD20+ B cells in lymphatic follicles of white pulp (arrow). E: combined filgrastim and rapamycin-treated group showing CD20+ B cells in lymphatic follicles of white pulp (arrow) (Immunoperoxidase technique for CD20 x 10, scale bar 50 um) TTA: thioacetamide

Supportive validation

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Experimental details: Fig. 6 UVRAG FS promotes spontaneous tumorigenesis in mice. a Kaplan-Meier plot of time to development of any malignancy in Dox-treated iUVRAG FS mice ( n = 25) versus Dox-treated control mice ( n = 25). Malignancy is determined by complete histologic survey of all major internal organs. *** P < 0.001 (Log-rank test). b Representative images of DLBCL in different organs that developed in Dox-treated iUVRAG FS mice. c Percentage of control ( n = 16) and UVRAG FS -expressing ( n = 12) mice with spontaneous tumors, including lymphoproliferative disease (LPD), lymphomas, and non-lymphoid malignancies. d H&E and IHC analysis of the most frequently observed neoplastic lesions from Dox-treated iUVRAG FS mice using the antibodies as indicated. Scale bars, 100 mum.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 3 IHC staining of an implant using fracture technique (20x magnification). ( a ) CD3; ( b ) CD20; ( c ) IgG. Upper (Blue) and lower (Maroon) region of interest are labelled. A/F--abutment-fixture junction.