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EuroMAbNet: European Monoclonal Antibodies Network

Antibody Identity card

Antibody information [TOX (Thymocyte Selection Associated High Mobility Group Box)]

TXRX10

Clone name TXRX10
Description Rat monoclonal
Antigen used Unknown
Epitope Unknown
Isotype IgG2a, kappa
Confirmed species reactivity Human
Ab used ThermoFisher Cat. 14-6502-82 (0.5 mg/ml)

APPLICATION	Recommended concentration	Status	Protocol
Western blotting (WB)	2.5 ug/ml	Working	Odyssey Western Blotting protocol (OdWB)
Immunocytochemistry (ICC)	5 ug/ml	Not working	ICC in frozen tissue and cytospin preparation
Immunohistochemistry (IHC-P)	5 μg/ml	Working	BOND-MAX automated immunohistochemistry
Immunoflourescence (IF)	25 ug/ml	Working	Immunofluorescence staining
Flow cytometry (FC)	5 ug/ml	Working	Flow cytometry
IHC-P Species	6.25 ug/ml	Working	BOND-MAX automated immunohistochemistry

Ab ID Card application validation / characterisation

WB Validation

Over-expression/cross reactivity

WB Validation: Over-expression/cross reactivity — To confirm the lack of cross-reactivity of the TXRX10 mAb with the other TOX family members, Western blotting (WB) was performed using cell extracts of MYC-FLAG tagged human TOX, MYC-tagged TOX2 and GFP-tagged TOX3 and TOX4. Two bands of 68 kDa and 57 kDa were detected only in the HEK-TOX cell extracts. Anti-GAPDH was used as loading control.

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Gene inactivation

WB Validation: Gene inactivation — The specificity of the TXRX10 mAb for the endogenous TOX protein was confirmed by WB using cell extracts of the MOLT4 cell line before and after TOX gene inactivation (MOLT4 KO TOX) using CRISPR-Cas9 technology. Two bands of ~68 kDa and ~57 kDa were observed only in the MOLT4 cell line. Anti-GAPDH was used as loading control.

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WB Characterisation

Endogenous expression

WB Characterisation: Endogenous expression — WB using the TXRX10 mAb showed 68 kDa and 57 kDa bands present in SUDHL4 diffuse large B-cell lymphoma line. TOX was not expressed in the U266 myeloma cell line. Anti-GAPDH was used as loading control.

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ICC / ICC-P Validation

Over-expression/cross reactivity

ICC Validation: Over-expression/cross reactivity — To confirm the lack of cross-reactivity with the other TOX family members, immunocytochemistry (ICC) with the TXRX10 mAb was performed on frozen cytospin preparations of MYC-tagged human TOX and TOX2 and GFP-tagged human TOX3 and TOX4 proteins expressed in HEK293T cells. TXRX10 mAb stained only the TOX transfectants. Labelling with anti-MYC and anti-GFP confirmed the efficiency of transfection.

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Gene inactivation

Not tested

IHC-P Characterisation

Endogenous expression

IHC-P Characterisation: Endogenous expression — Single immunoperoxidase labelling of tonsil with the TXRX10 mAb shows strong nuclear staining of TOX in germinal centre cells and cells in the interfollicular area. (B) TOX is also highly expressed by cells in the thymic cortex. (C) In spleen, TOX is expressed by scattered cells in the white and red pulp.

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IF Characterisation

Endogenous expression

IF Characterisation: Endogenous expression — Triple immunofluorescence staining of human paraffin embedded tonsil with the TXRX10 mAb (green), anti-CD20 (red) and anti-CD3 (white).

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FC Validation

Over-expression/cross reactivity

FC Validation: Over-expression/cross reactivity — To confirm the lack of cross-reactivity with the other TOX family members, flow cytometry with TXRX10 mAb was performed on wild type HEK293T cells (dark green) and HEK293T cell lines transfected with MYC-tagged human TOX (light green) and TOX2 (orange) and GFP-tagged TOX3 (light blue) and TOX4 (red) plasmid vectors.

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Gene inactivation

FC Validation: Gene inactivation — The specificity of TXRX10 mAb for the detection of endogenous TOX protein was confirmed by flow cytometry using the MOLT4 cell line before (red) and after TOX gene inactivation (MOLT4 KO TOX, blue) using CRISPR-Cas9 technology.

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FC Characterisation

Endogenous expression

FC Characterisation: Endogenous expression — Flow cytometry using the TXRX10 mAB shows endogenous TOX protein in the MOLT4 cells (red) but not in the U266 cells (blue).

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IHC-P Domestic species

Canary Serinus canaria: +
Cat Felis catus: -
Cow Bos taurus: +++
Dog Canis lupus familiaris: +++
Goat Capra aegagrus: +++
Horse Equus ferus: ++
Jacobin Pigeon Columba livia: ++
Llama Llama glama: +++
Mouse Mus musculus: +++
Pig Sus scrofa domesticus: +++
Rat Rattus norvegicus: -
Sheep Ovis orientalis: +++

IHC-P Domestic species — Immunoperoxidase staining of TXRX10 mAb in paraffin sections of lymph node from a variety of domestic animal species.

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IHC-P Wild species

African sacred ibis Threskiornis aethiopicus: +
Asian black bear Ursus thibetanus: ++
Baboon Papio sp.: +++
Badger Meles meles: -
Bush Dog Speothos venaticus: ++
Common brown lemur Eulemur fulvus mayottensis: -
Emu Dromaius novaehollandiae: -
European bison Bison bonasus: ++
Mandrill Mandrillus sphinx: +++
Otter Pteronura brasiliensis: -
Patagonian mara Dolichotis patagonum: +++
Penguin Spheniscus demersus: -
Raccoon Procyon lotor: +++
Red flamimgo Phoenicopterus ruber: ++
Reindeer Rangifer tarandus: +++
Rhea Rhea americana: -
Sandbar shark Carcharhinus plumbeus: -
Sitatunga Tragelaphus spekii: ++
South American sea lion Otaria byronia: +
Stork Ciconia ciconia: -
Wild pig Sus scrofa: +
Wolf Canis lupus: +

IHC-P Wild species — Immunoperoxidase staining with the TXRX10 mAb of paraffin sections of lymph node from a variety of wild exotic animal species.

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