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EuroMAbNet: European Monoclonal Antibodies Network

Antibody Identity card

Antibody information [CD1d ]

SNOW631C

Clone name SNOW631C
Description Rat monoclonal
Antigen used RBL1-CD1d transfected cells and CD1d-HIS
Epitope GTFSDQQWETLQHIF
Isotype IgG2a
Confirmed species reactivity Human
Ab used CNIO purified antibody (3.4mg/ml)

APPLICATION	Recommended concentration	Status	Protocol
Western blotting (WB)	Neat tissue culture supernatant	Working	Western Blotting (WB)
Immunocytochemistry (ICC)	Neat tissue culture supernatant	Working	ICC in frozen tissue and cytospin preparation
Immunohistochemistry (IHC-P)	250ug/ml	Working	BOND-MAX automated immunohistochemistry
Immunoflourescence (IF)	250ug/ml	Working	Immunofluorescence staining
Flow cytometry (FC)	0.22 mg/ml	Working	Flow cytometry
IHC-P Species		Not tested

Ab ID Card application validation / characterisation

WB Validation

Over-expression/cross reactivity

CD1d antibody WB Validation: Over-expression/cross reactivity — To confirm the lack of cross-reactivity with the other CD1 family members, Western blotting (WB) of the SNOW631C mAb was performed using cell extracts of HEK293T transfectants expressing CD1a, CD1b, CD1c, CD1d or CD1e. A 50 kDa band was detected by WB only in the HEK-CD1d cell extract. Anti-vinculin was used as loading control

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Gene inactivation

CD1d antibody WB Validation: Gene inactivation — The specificity of the SNOW631C mAb for the endogenous CD1d protein was confirmed by WB using cell extracts of Jurkat cell line before (JURKAT WT) and after CD1d gene inactivation (CD1D KO JURKAT) using CRISPR-Cas9 technology. A 50 kD band was observed only in the JURKAT WT cells. Anti-vinculin was used as loading control.

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WB Characterisation

Endogenous expression

CD1d antibody WB Characterisation: Endogenous expression — WB using the SNOW631C mAb showed 50 kDa bands of CD1d protein in the MOLT4 (lymphoblastic leukaemia) and thymus lysates. CD1d was not expressed in the VL51 (splenic B-cell lymphoma), U266, RPMI8226 (myeloma) and Ramos (Burkitt's lymphoma) cell lines. Anti-vinculin was used as a loading control.

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ICC / ICC-P Validation

Over-expression/cross reactivity

CD1d antibody ICC Validation: Over-expression/cross reactivity — To confirm lack of cross-reactivity with the other CD1 family members, immunocytochemistry (ICC) with the SNOW631C mAb was performed on frozen cytospin preparations of MYC-tagged CD1a, CD1b and CD1d as well as FLAG-tagged CD1c and GFP-tagged CD1e proteins expressed in HEK293T transfectants. Staining was observed only in the CD1d transfectants. Labelling with the anti-MYC, anti-FLAG and anti-GFP confirmed the efficiency of transfection.

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Gene inactivation

CD1d antibody ICC Validation: Gene inactivation — The specificity of the SNOW631C mAb for the endogenous CD1d protein was confirmed by ICC in sections from formalin fixed paraffin embedded pellets of the JURKAT cell line before and after (CD1d KO JURKAT) gene inactivation using CRISPR-Cas9 technology. CD1d expression was observed in wild type JURKAT cells, while no staining was observed in the JURKAT Cd1d KO cells.

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IHC-P Characterisation

Endogenous expression

CD1d antibody IHC-P Characterisation: Endogenous expression — Single immunoperoxidase labelling with the SNOW631C mAb demonstrated cytoplasmic and membrane staining of the CD1d protein in the mantle zone surrounding the germinal centre in the tonsil. In the thymus, strong staining was observed in the cortex, whereas fewer stained cells were observed in the medulla. In bone marrow an unexpected labelling of erythroblasts was observed. In the spleen, CD1d was expressed in B cells within the mantle and marginal zone cells.

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IF Characterisation

Endogenous expression

CD1d antibody IF Characterisation: Endogenous expression — Double immunofluorescence staining of human paraffin embedded thymus by the SNOW631C mAb (red) and anti-CD8 (green).

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FC Validation

Over-expression/cross reactivity

Not working

Gene inactivation

Not working

FC Characterisation

Endogenous expression

Not working

IHC-P Domestic species

Not tested

IHC-P Wild species

Not tested

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