B7-H3/CD276 Antibody (3108D) [Unconjugated]

B7-H3 was detected in immersion fixed paraffin-embedded sections of human Lung Tumor using Rabbit Anti-Human B7-H3, Monoclonal Antibody (Catalog #MAB11721) at 1ug/mL at 37 ° Celsius for 4 minutes. Before incubation with the primary antibody, tissue underwent an all-in-one dewaxing and antigen retrieval preprocessing using PreTreatment Module (PT Module) and Dewax and HIER Buffer H (pH 9; Epredia Catalog # TA-999-DHBH). Tissue was stained using the Alexa Fluor™ Plus 647 Goat anti-Rabbit IgG Secondary Antibody at 1:200 at 37 ° Celsius for 2 minutes. (Yellow; Lunaphore Catalog # DR647RB) and counterstained with DAPI (blue; Lunaphore Catalog # DR100). Specific staining was localized to the membrane. Protocol available in COMET™ Panel Builder.

B7‑H3 was detected in immersion fixed paraffin-embedded sections of human liver cancer using Rabbit Anti-Human B7‑H3 Monoclonal Antibody (Catalog # MAB11721) at 0.5 µg/ml for 1 hour at room temperature followed by incubation with the Anti-Rabbit IgG VisUCyte™ HRP Polymer Antibody (Catalog # VC003) or the HRP-conjugated Anti-Rabbit IgG Secondary Antibody (Catalog # HAF008). 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 the membrane. View our protocol for IHC Staining with VisUCyte HRP Polymer Detection Reagents.

B7‑H3 was detected in immersion fixed paraffin-embedded sections of human lung cancer using Rabbit Anti-Human B7‑H3 Monoclonal Antibody (Catalog # MAB11721) at 0.5 µg/ml for 1 hour at room temperature followed by incubation with the Anti-Rabbit IgG VisUCyte™ HRP Polymer Antibody (Catalog # VC003) or the HRP-conjugated Anti-Rabbit IgG Secondary Antibody (Catalog # HAF008). 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 the membrane. View our protocol for IHC Staining with VisUCyte HRP Polymer Detection Reagents.

Western Blot shows lysates of JAR human choriocarcinoma cell line and LNCaP human prostate cancer cell line. PVDF membrane was probed with 0.25 µg/ml of Rabbit Anti-Human B7‑H3 Monoclonal Antibody (Catalog # MAB11721) followed by HRP-conjugated Anti-Rabbit IgG Secondary Antibody (Catalog # HAF008). A specific band was detected for B7‑H3 at approximately 90-110 kDa (as indicated). This experiment was conducted under reducing conditions and using Western Blot Buffer Group 1.

Left: Simple Western lane view shows lysates of LNCaP human prostate cancer cell line, loaded at 0.1 mg/ml. A specific band was detected for B7‑H3 at approximately 149 kDa (as indicated) using both 2 µg/ml and 10 µg/ml of Rabbit Anti-Human B7‑H3 Monoclonal Antibody (Catalog # MAB11721) followed by HRP-conjugated Goat Anti-Rabbit Secondary Antibody (Catalog # 042-206). This experiment was conducted under reducing conditions and using the 12-230kDa separation system. Right: Simple Western electropherogram showing the same Rabbit Anti-Human B7‑H3 Monoclonal Antibody (Catalog # MAB11721) tested at 2 µg/ml (blue line) and 10 µg/ml (green line) in the LNCaP human prostate cancer cell line.

• Reactivity: Hu
• Applications: WB, Simple Western, IHC, mIF
• Clone: 3108D
• Clonality: Monoclonal
• Host: Rabbit
• Conjugate: Unconjugated

B7-H3/CD276 Antibody (3108D) [Unconjugated] Summary

• Additional Information: Recombinant Monoclonal Antibody.
• Immunogen: Mouse myeloma cell line, NS0-derived human B7-H3
  Leu29-Pro245
  Accession # Q5ZPR3
• Specificity: Detects recombinant human B7-H3 protein in Direct ELISA.
• Source: N/A
• Isotype: IgG
• Clonality: Monoclonal
• Host: Rabbit
• Purity Statement: Protein A or G purified from hybridoma culture supernatant

Applications/Dilutions

• Dilutions:
  • Immunohistochemistry 0.5-10 ug/mL
  • Multiplex Immunofluorescence 1 ug/mL
  • Simple Western 2-10 ug/mL
  • Western Blot 0.25 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.
• Reconstitution Instructions: Reconstitute lyophilized material at 0.2 mg/ml in sterile PBS. For liquid material, refer to CoA for concentration.

Notes

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

Alternate Names for B7-H3/CD276 Antibody (3108D) [Unconjugated]

• B7H3
• B7-H3
• B7H34Ig-B7-H3
• B7-H3B7 homolog 3
• CD276 antigen
• CD276 molecule
• CD276
• Costimulatory molecule

Background

Human B7 homolog 3 (B7-H3) is a member of the B7 family of immune proteins that provide signals for regulating immune responses (1‑3). Other family members include B7-1, B7-2, B7-H2, PD-L1 (B7-H1), and PD-L2. B7 proteins are immunoglobulin (Ig) superfamily members with extracellular Ig-V-like and Ig-C-like domains and short cytoplasmic domains. Among the family members, they share about 20‑40% amino acid (aa) sequence identity. The cloned human B7-H3 cDNA encodes a 316 aa type I membrane precursor protein with a putative 28 aa signal peptide, a 217 aa extracellular region containing one V-like and one C-like Ig domain, a transmembrane region, and a 45 aa cytoplasmic domain. An isoform of human B7-H3 containing a four-Ig-like domain extracellular region has also been identified. Human B7-H3 is not expressed on resting B cells, T cells, monocytes or dendritic cells, but is induced on dendritic cells and monocytes by inflammatory cytokines. B7-H3 expression is also detected on various normal tissues and in some tumor cell lines. Human B7-H3 does not bind any known members of the CD28 family of immunoreceptors. However, B7-H3 has been shown to bind an unidentified counter-receptor on activated T cells to costimulate the proliferation of CD4+ or CD8+ T cells. B7-H3 has also been found to enhance the induction of primary cytotoxic T lymphocytes and stimulate IFN-gamma production (1‑3).

1. Chapoval, A.I. et al. (2001) Nat. Immunol. 2:269.
2. Sharpe, A.H. and G.J. Freeman (2002) Nat. Rev. Immunol. 2:116.
3. Coyle, A. and J. Gutierrez-Ramos (2001) Nat. Immunol. 2:203.

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.