Hello, I want to do IHC with LAG3 antibody, but I don’t know how to choose the positive control tissue or cell line. Can you help?

Name: Human LAG-3 Antibody
Brand: R&D Systems
SKU: MAB231971

LAG-3 protein is expressed in activated NK and T-lymphocytes. As such, finding a good tissue or cell line for positive control testing may be difficult. Some of the papers linked below may offer some help: https://pathsocjournals.onlinelibrary.wiley.com/doi/abs/10.1002/path.1679 http://clincancerres.aacrjournals.org/content/21/17/3969.full.pdf https://www.researchgate.net/profile/Devinder_Gill2/publication/7024802_Expression_of_LAG-3_by_tumor-infiltrating_lymphocytes_is_coincident_with_the_suppression_of_latent_membrane_antigen-specific_CD8_T-cell_function_in_Hodgkin_lymphoma_patients/links/0fcfd50bfccc52342f000000.pdf http://cancerres.aacrjournals.org/content/75/15_Supplement/271.short For some data for normal tissue and cell line expression of the LAG-3 protein, you can check the Human Protein Atlas site. For tissues, appendix, lymph node, and tonsil tissue seem to show the best level of expression for this protein: http://www.proteinatlas.org/ENSG00000089692-LAG3/tissue. With regard to cell lines, the HDLM-2 cell line by far has the best level of expression: http://www.proteinatlas.org/ENSG00000089692-LAG3/cell/HPA013967. I hope this data will help you to select a good control for your experiment. Please let us know if you have any additional questions.

Human LAG-3 Antibody

R&D Systems, part of Bio-Techne | Catalog # MAB231971

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Product Specifications
Scientific Data
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Preparation & Storage
Background
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Key Product Details

Species Reactivity

Human

Applications

Immunocytochemistry, Immunohistochemistry

Label

Unconjugated

Antibody Source

Monoclonal Mouse IgG₁ Clone # 1058221

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Product Specifications

Immunogen

Human Lag-3 Peptide

Accession # P18627

Specificity

Detects human LAG-3 in direct ELISA.

Clonality

Monoclonal

Host

Mouse

Isotype

IgG₁

Scientific Data Images for Human LAG-3 Antibody

Detection of LAG-3 in tonsil.

LAG-3 was detected in immersion fixed paraffin-embedded sections of tonsil using Mouse Anti-Human LAG-3 Monoclonal Antibody (Catalog # MAB231971) at 5 µg/mL for 1 hour at room temperature followed by incubation with the Anti-Mouse IgG VisUCyte™ HRP Polymer Antibody (Catalog # VC001). Before incubation with the primary antibody, tissue was subjected to heat-induced epitope retrieval using VisUCyte Antigen Retrieval Reagent-Basic (Catalog # VCTS021). Tissue was stained using DAB (brown) and counterstained with hematoxylin (blue). Specific staining was localized to lymphocytes. View our protocol for IHC Staining with VisUCyte HRP Polymer Detection Reagents.

LAG-3 in HDLM-2 (Positive) & Daudi (Negative).

LAG-3 was detected in immersion fixed HDLM-2 Human Hodgkin’s lymphoma cell line (Positive) & Daudi Human Burkitt's lymphoma cell line (Negative) using Mouse Anti-Human LAG-3 Monoclonal Antibody (Catalog # MAB231971) at 8 µg/mL for 3 hours at room temperature. Cells were stained using the NorthernLights™ 557-conjugated Anti-Mouse IgG Secondary Antibody (red; Catalog # NL007) and counterstained with DAPI (blue). Specific staining was localized to cytoplasm. View our protocol for Fluorescent ICC Staining of Non-adherent Cells.

Applications for Human LAG-3 Antibody

Application

Recommended Usage

Immunocytochemistry

8-25 µg/mL
Sample: Immersion fixed HDLM-2 human Hodgkin's lymphoma cell line (Positive) & Daudi human Burkitt's lymphoma cell line (Negative)

Immunohistochemistry

5-25 µg/mL
Sample: Immersion fixed paraffin-embedded sections of tonsil

Formulation, Preparation, and Storage

Purification

Protein A or G purified from hybridoma culture supernatant

Reconstitution

Reconstitute at 0.5 mg/mL in sterile PBS. For liquid material, refer to CoA for concentration.

Formulation

Lyophilized from a 0.2 μm filtered solution in PBS with Trehalose. *Small pack size (SP) is supplied either lyophilized or as a 0.2 µm filtered solution in PBS.

Shipping

Lyophilized product is shipped at ambient temperature. Liquid small pack size (-SP) is shipped with polar packs. Upon receipt, store immediately at the temperature recommended below.

Stability & Storage

Use a manual defrost freezer and avoid repeated freeze-thaw cycles.

12 months from date of receipt, -20 to -70 °C as supplied.
1 month, 2 to 8 °C under sterile conditions after reconstitution.
6 months, -20 to -70 °C under sterile conditions after reconstitution.

Background: LAG-3

LAG-3 (Lymphocyte activation gene-3), also known as CD223, is a member of the immunoglobulin superfamily (IgSF). The mature LAG-3 protein is a 496 amino acid (aa) membrane protein with a 421 aa extracellular region which contains four IgSF domains, a 21 aa transmembrane region and a 54 aa cytoplasmic region. LAG-3 and CD4 molecules share <20% aa sequence homology but have a similar structure (1, 2). Both molecules bind to MHC class II. LAG-3 binds to MHC class II with higher affinity compared to CD4. Both LAG-3 and CD4 genes are located on the distal part of the short arm of chromosome 12. LAG-3 is an activation-induced molecule, expressed on activated T cells and NK cells, but not on resting T cells. Studies using LAG-3 -/- mice have shown significant delay of T cell apoptosis following antigen stimulation and increased size of memory T cells pool following infection (3, 4). It also has been reported that anti-LAG-3 antibodies up-regulate T cell activation by blocking interaction of LAG-3 and MHC class II. The study has demonstrated that LAG-3 is selectively expressed on activated CD4+CD25+ TReg cells and plays a role in their suppressive activity (5). This evidence indicated, unlike the interaction of CD4 with MHC class II that plays a positive role in T cell activation, LAG-3 binds to MHC class II and negatively regulates T cell activation through LAG-3 signaling. On the other hand, studies have shown that binding of LAG-3 to MHC class II molecules on antigen presenting cells induce maturation of dendritic cells and cytokine secretion by monocytes through MHC class II signal transduction (6). Taken together, LAG-3 may have two major functions, it negatively regulates T cells activation through LAG-3 signaling and stimulates antigen presenting cells which express MHC class II.

References

Triebel, F. et al. (1990) J. Exp. Med. 171:1393.
Baixeras, E. et al. (1992) J. Exp. Med 176:327.
Workman, C.J. and D.A. Vignali (2003) Eur. J. Immunol. 33:970.
Workman, C.J. et al. (2004) J. Immunol. 172:5450.
Huang, C.T. et al. (2004) Immunity 21:503.
Andreae, S. et al. (2003) Blood 102:2130.

Long Name

Lymphocyte-activation Gene 3

Alternate Names

CD223, LAG3

Entrez Gene IDs

3902 (Human); 16768 (Mouse); 297596 (Rat); 102122272 (Cynomolgus Monkey)

Gene Symbol

LAG3

UniProt

P18627

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Product Specific Notices for Human LAG-3 Antibody

For research use only

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