TIM-3 Antibody [Unconjugated]

Western blot shows lysates of RAW 264.7 mouse monocyte/macrophage cell line. PVDF membrane was probed with 2 µg/mL of Goat Anti-Mouse TIM-3 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1529) followed by HRP-conjugated Anti-Goat IgG Secondary Antibody (Catalog # HAF017). A specific band was detected for TIM-3 at approximately 45-70 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.

TIM‑3 was detected in immersion fixed paraffin-embedded sections of mouse spleen using Goat Anti-Mouse TIM‑3 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1529) at 3 µg/mL for 1 hour at room temperature followed by incubation with the Anti-Goat IgG VisUCyte™ HRP Polymer Antibody (VC004). Before incubation with the primary antibody, tissue was subjected to heat-induced epitope retrieval using Antigen Retrieval Reagent-Basic (CTS013). Tissue was stained using DAB (brown) and counterstained with hematoxylin (blue). Specific staining was localized to lymphocytes. Staining was performed using our protocol for IHC Staining with VisUCyte HRP Polymer Detection Reagents.

Blocking TIM-3 significantly reduces brain injury after hypoxia-ischaemia.(a) Representative images of TTC-stained brain slices from H/I mice treated with 100 μg of IgG (n=12) or anti-TIM-3 antibody (n=12). The infarct volume was quantified with Image J analyser and expressed as a percentage of the damaged ipsilateral hemisphere. (b) Representative magnetic resonance images (MRIs) from TIM-3-antibody-treated mice (n=4) and IgG-treated mice (n=4) at 24 h post-H/I. (c) Representative T2 images from TIM-3-antibody-treated mice (n=4) and IgG-treated mice (n=4) after H/I. (d) The extent of the oedema formation was obtained from the T2-weighted MRI images and ADC map. (e) Representative confocal microscopic images of immunohistochemical staining for NeuN and cleaved caspase-3 in coronal brain sections from IgG- and anti-TIM-3-treated H/I mice 24 h after injury. Scale bar, 50 μm. The graph shows the mean number of NeuN and cleaved caspase-3-stained cells per mm2. (f) Immunoblot detection of full-length PARP proteins in contralateral and ipsilateral cortex regions of control IgG- or anti-TIM-3-treated mice. The graph shows the relative levels of full-length PARP (116 kDa). Data represent the mean±s.d. from at least three independent experiments. Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/25790768), licensed under a CC-BY license. Not internally tested by R&D Systems.

Blocking TIM-3 significantly reduces brain injury after hypoxia-ischaemia.(a) Representative images of TTC-stained brain slices from H/I mice treated with 100 μg of IgG (n=12) or anti-TIM-3 antibody (n=12). The infarct volume was quantified with Image J analyser and expressed as a percentage of the damaged ipsilateral hemisphere. (b) Representative magnetic resonance images (MRIs) from TIM-3-antibody-treated mice (n=4) and IgG-treated mice (n=4) at 24 h post-H/I. (c) Representative T2 images from TIM-3-antibody-treated mice (n=4) and IgG-treated mice (n=4) after H/I. (d) The extent of the oedema formation was obtained from the T2-weighted MRI images and ADC map. (e) Representative confocal microscopic images of immunohistochemical staining for NeuN and cleaved caspase-3 in coronal brain sections from IgG- and anti-TIM-3-treated H/I mice 24 h after injury. Scale bar, 50 μm. The graph shows the mean number of NeuN and cleaved caspase-3-stained cells per mm2. (f) Immunoblot detection of full-length PARP proteins in contralateral and ipsilateral cortex regions of control IgG- or anti-TIM-3-treated mice. The graph shows the relative levels of full-length PARP (116 kDa). Data represent the mean±s.d. from at least three independent experiments. Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/25790768), licensed under a CC-BY license. Not internally tested by R&D Systems.

Blocking TIM-3 significantly reduces brain injury after hypoxia-ischaemia.(a) Representative images of TTC-stained brain slices from H/I mice treated with 100 μg of IgG (n=12) or anti-TIM-3 antibody (n=12). The infarct volume was quantified with Image J analyser and expressed as a percentage of the damaged ipsilateral hemisphere. (b) Representative magnetic resonance images (MRIs) from TIM-3-antibody-treated mice (n=4) and IgG-treated mice (n=4) at 24 h post-H/I. (c) Representative T2 images from TIM-3-antibody-treated mice (n=4) and IgG-treated mice (n=4) after H/I. (d) The extent of the oedema formation was obtained from the T2-weighted MRI images and ADC map. (e) Representative confocal microscopic images of immunohistochemical staining for NeuN and cleaved caspase-3 in coronal brain sections from IgG- and anti-TIM-3-treated H/I mice 24 h after injury. Scale bar, 50 μm. The graph shows the mean number of NeuN and cleaved caspase-3-stained cells per mm2. (f) Immunoblot detection of full-length PARP proteins in contralateral and ipsilateral cortex regions of control IgG- or anti-TIM-3-treated mice. The graph shows the relative levels of full-length PARP (116 kDa). Data represent the mean±s.d. from at least three independent experiments. Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/25790768), licensed under a CC-BY license. Not internally tested by R&D Systems.

TIM-3 is highly expressed in hypoxic brain regions of a H/I mouse model.(a) TIM-3 transcript levels were examined in brain tissues from the contralateral cortex (C, boxed region) and ischaemic ipsilateral cortex (I, boxed region) of mouse model 24 h after H/I. The RT–PCR products were quantified with Image J and normalized with respect to the expression of actin. The HIF-1 alpha transcript level represents a positive control for hypoxia. The right panel shows representative TTC staining of three brain sections from the H/I mice. (b) Representative western blot analyses of the TIM-3 and HIF-1 alpha proteins (n=3). Relative levels of TIM-3 are shown as the mean±s.d. from three independent experiments. (c) Contralateral and ipsilateral cortical regions of coronal sections from the H/I mice were subjected to immunohistochemistry using an anti-TIM-3 antibody, and the number of TIM-3-expressing cells per mm2 was counted. (d) Immunohistochemistry was performed on brain sections from the H/I mice using anti-TIM-3 and hypoxyprobe-1 (red, to detect hypoxic regions). Scale bars, 50 μm ( × 20); 50 μm ( × 40). (e,f) Brain cells were isolated from the ipsilateral and contralateral hemispheres of three mice per group, processed for simultaneous detection of TIM-3 plus Iba-1 (e) or GFAP (f), and analysed by FACS. The results are presented as relative TIM-3 levels in the indicated gated populations, as determined from three independent experiments. Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/25790768), licensed under a CC-BY license. Not internally tested by R&D Systems.

• Reactivity: Mu
• Applications: WB, Flow, IHC, CyTOF-ready
• Clonality: Polyclonal
• Host: Goat
• Conjugate: Unconjugated
• Concentration: LYOPH

TIM-3 Antibody [Unconjugated] Summary

• Immunogen: Mouse myeloma cell line NS0-derived recombinant mouse TIM‑3
  Leu22-Arg191
  Accession # AAL65156
• Specificity: Detects mouse TIM-3 in direct ELISAs and Western blots. In direct ELISAs, approximately 10% cross-reactivity with recombinant human TIM‑3 is observed and less than 5% cross-reactivity with recombinant mouse (rm) TIM‑1, rmTIM‑2, rmTIM‑4, rmTIM‑5, rmTIM‑6, and rmTIM‑7 is observed.
• Source: N/A
• Isotype: IgG
• Clonality: Polyclonal
• Host: Goat
• Gene: HAVCR2
• Purity Statement: Antigen Affinity-purified

Applications/Dilutions

• Dilutions:
  • CyTOF-ready
  • Flow Cytometry 2.5 ug/10^6 cells
  • Immunohistochemistry 3-15 ug/mL
  • Western Blot 2 ug/mL

Packaging, Storage & Formulations

• 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.
• Buffer: 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.
• Preservative: No Preservative
• Concentration: LYOPH
• Reconstitution Instructions: Reconstitute at 0.2 mg/mL in sterile PBS.

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for TIM-3 Antibody [Unconjugated]

• CD366
• FLJ14428
• HAVCR2
• HAVcr-2
• hepatitis A virus cellular receptor 2
• kidney injury molecule-3
• KIM-3
• SPTCL
• T cell immunoglobulin mucin 3
• T cell immunoglobulin mucin-3
• T-cell immunoglobulin and mucin domain-containing protein 3
• TIM 3
• TIM3 T-cell membrane protein 3
• TIM3
• TIM-3
• TIMD3
• TIMD-3
• TIMD3KIM-3

Background

TIM-3 (T cell immunoglobulin and mucin domain-3) is a 60 kDa member of the TIM family of immune regulating molecules. TIMs are type I transmembrane glycoproteins with one Ig-like V-type domain and a Ser/Thr-rich mucin stalk (1-3). There are three TIM genes in human and eight in mouse. Mature mouse TIM-3 consists of a 174 amino acid (aa) extracellular domain (ECD), a 21 aa transmembrane segment (TM), and a 67 aa cytoplasmic tail (4). Two alternately spliced isoforms have been reported in mouse which lack either the TM or both the TM and mucin regions (5, 6). Within the ECD, mouse TIM-3 shares 58% and 74% aa sequence identity with human and rat TIM-3, respectively. TIM-3 is specifically expressed on Th1 cells whereas TIM-1 and TIM-2 are expressed on Th2 cells. In chronic inflammation, autoimmune disorders, and some cancers, TIM-3 is upregulated on several other hematopoietic cell types and on hippocampal neurons (9-12). The glycosylated Ig domain of TIM-3 binds cell-associated galectin-9 which induces TIM-3 Tyr phosphorylation and proapoptotic signaling (10, 13). TIM-3 functions as a negative regulator of Th1 cell activity. Its blockade results in increased IFN-gamma production, Th1 cell proliferation, and cytotoxicity (5, 7, 12, 14). TIM-3 may play a role in regulatory T cell development (7), inflammation (15), and immune tolerance (5, 13, 14). Soluble mouse TIM-3 has been shown to inhibit anti-tumor effector T cell responses and to enhance autoimmune reactions (6, 7).

1. Anderson, A.C. and D.E. Anderson (2006) Curr. Opin. Immunol. 18:665.
2. Mariat, C. et al. (2005) Phil. Trans. R. Soc. B 360:1681.
3. Meyers, J.H. et al. (2005) Trends Mol. Med. 11:362.
4. Monney, L. et al. (2002) Nature 415:536.
5. Sabatos, C.A. et al. (2003) Nat. Immunol. 4:1102.
6. Geng, H. et al. (2006) J. Immunol. 176:1411.
7. Sanchez-Fueyo, A. et al. (2003) Nat. Immunol. 4:1093.
8. Khademi, M. et al. (2004) J. Immunol. 172:7169.
9. Wiener, Z. et al. (2007) J. Invest. Dermatol. 127:906.
10. van de Weyer, P.S. et al. (2006) Biochem. Biophys. Res. Commun. 351:571.
11. Gielen, A.W. et al. (2005) J. Neuroimmunol. 164:93.
12. Oikawa, T. et al. (2006) J. Immunol. 177:4281.
13. Zhu, C. et al. (2005) Nat. Immunol. 6:1245.
14. Koguchi, K. et al. (2006) J. Exp. Med. 203:1413.
15. Frisancho-Kiss, S. et al. (2006) J. Immunol. 176:6411.

Limitations

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Primary Antibodies are guaranteed for 1 year from date of receipt.

Publications for TIM-3 Antibody (AF1529)(4)

Publications using AF1529

• E Wingrove, ZZ Liu, KD Patel, A Arnal-Esta, WL Cai, MA Melnick, K Politi, C Monteiro, L Zhu, M Valiente, HM Kluger, VL Chiang, DX Nguyen Transcriptomic Hallmarks of Tumor Plasticity and Stromal Interactions in Brain Metastasis Cell Rep, 2019-04-23;27(4):1277-1292.e7. 2019-04-23 [PMID: 31018140] (IHC, Mouse)
• Elk Kossatz, Rafael Maldonado, Patricia Robledo CB2 cannabinoid receptors modulate HIF-1 alpha and TIM-3 expression in a hypoxia-ischemia mouse model European Neuropsychopharmacology 2016-12-01 [PMID: 28253997] (Immunohistochemistry, Transgenic Mouse)
• Koh H, Chang C, Jeon S, Yoon H, Ahn Y, Kim H, Kim I, Jeon S, Johnson R, Park E The HIF-1/glial TIM-3 axis controls inflammation-associated brain damage under hypoxia. Nat Commun, 2015-03-20;6(0):6340. 2015-03-20 [PMID: 25790768] (ICC, Mouse)
• Nakae S, Iikura M, Suto H, Akiba H, Umetsu DT, DeKruyff RH, Saito H, Galli SJ TIM-1 and TIM-3 enhancement of Th2 cytokine production by mast cells. Blood, 2007-07-09;110(7):2565-8. 2007-07-09 [PMID: 17620455] (Western Blot, Mouse)

Bioinformatics

• Gene Symbol: HAVCR2
• Uniprot:
  • AAL65156()