Histology, immunohistochemistry and quantitation was performed as described previously [13 (link),28 (link),46 (link)]. Briefly, feet were fixed in paraformadehyde, decalcified and embedded in paraffin, and sections stained with hematoxylin and eosin (H&E). For immunohistochemistry, sections were stained with anti-NKp46 (rabbit polyclonal; Biorybt, Berkeley, CA) or anti-CD3 (A0452; Dako, North Sydney, Australia), with detection using MACH 2 (Biocare, Concord, CA) and Nova Red. F4/80 staining was undertaken as described [28 (link)]. Sections were scanned using Aperio AT Turbo (Aperio, Vista, CA) and analyzed using Aperio ImageScope software (v10) and the Positive Pixel Count v9 algorithm.
Anti cd3 a0452
Manufactured by Agilent Technologies
Sourced in Australia, United States
The Anti-CD3 (A0452) is a laboratory reagent produced by Agilent Technologies. It is an antibody that specifically binds to the CD3 cell surface receptor, which is a component of the T-cell receptor complex. This product is intended for research use only.
Automatically generated - may contain errors
Lab products found in correlation
3 protocols using anti cd3 a0452
1
Histologic and Immunohistochemical Analysis
2
Comprehensive miRNA and Immunohistochemistry Protocol
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miR ISH was performed as previously described [13 (link)]. Tumor sections were hybridized with double-DIG-LNA probes for miR-574-5p (38674-15) and miR-1246 (21239-15), and positive and negative control (Exiqon, Vedbaek, Denmark). Immunohistochemistry was carried out using an automated immunostainer (BenchMark Ultra, Ventana Medical Systems, Tucson, AZ, USA) with anti-CD3 (A0452) and anti-CD20 antibodies (M0755) purchased from Dako (Glostrup, Denmark).
3
Mutant NPM1 Protein Detection in Bone Marrow
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Immunohistochemistry to interrogate for mutant NPM1 protein (ThermoFisher Scientific, Rockford, IL; Catalog #: PA1-46356) was performed on 4-μm-thick BFPE sections from all diagnostic bone marrow biopsy samples using previouslyestablished protocols [16] . Visual scoring to determine the percentage of positively-staining cells in each case was performed independently by two board-certified hematopathologists (S.S.P. and O.K.W.) and results were averaged.
Multiplexed immunofluorescence (mIF) was performed by staining 4-μm-thick BFPE bone marrow core biopsy sections in a BondRX automated stainer, using published protocols [17] . One panel of primary antibody/fluorophore pairs was applied to all cases (listed in order of application sequence):
(1) anti-CD3 (A0452, 1:750, Dako), (2) anti-CD8 (C8/144B, 1:7000, Abcam), (3) anti-CD4 (4B12, 1:250, Invitrogen), (4) anti-CD34 (QBEnd/10, 1:10,500, BioLegend), (5) anti-Granzyme B (GrB-7, 1:100, Invitrogen), and (6) anti-mutant NPM1-specific antibody (as above, 1:2500). Antibody/Opal fluor combinations were utilized as follows: CD3/650 (1:100), CD8/540 (1:100), CD4/520 (1:100), CD34/570 (1:200), Granzyme B/620 (1:200), and anti-mutant NPM1/690 (1:50). All slides were also stained with 4′,6-diamidino-2-phenylindole (DAPI) for nuclear identification.
Multiplexed immunofluorescence (mIF) was performed by staining 4-μm-thick BFPE bone marrow core biopsy sections in a BondRX automated stainer, using published protocols [17] . One panel of primary antibody/fluorophore pairs was applied to all cases (listed in order of application sequence):
(1) anti-CD3 (A0452, 1:750, Dako), (2) anti-CD8 (C8/144B, 1:7000, Abcam), (3) anti-CD4 (4B12, 1:250, Invitrogen), (4) anti-CD34 (QBEnd/10, 1:10,500, BioLegend), (5) anti-Granzyme B (GrB-7, 1:100, Invitrogen), and (6) anti-mutant NPM1-specific antibody (as above, 1:2500). Antibody/Opal fluor combinations were utilized as follows: CD3/650 (1:100), CD8/540 (1:100), CD4/520 (1:100), CD34/570 (1:200), Granzyme B/620 (1:200), and anti-mutant NPM1/690 (1:50). All slides were also stained with 4′,6-diamidino-2-phenylindole (DAPI) for nuclear identification.
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