CD33 was detected in immersion fixed paraffin-embedded sections of human tonsil using Mouse Anti-Human CD33, Monoclonal Antibody (Catalog # MAB11371) at 15ug/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™ 647 Goat anti-Mouse IgG Secondary Antibody at 1:200 at 37 ° Celsius for 2 minutes. (Yellow; Lunaphore Catalog # DR647MS) and counterstained with DAPI (blue; Lunaphore Catalog # DR100). Specific staining was localized to the membrane. Protocol available in COMET™ Panel Builder.

CD33 in Human Thymus via seqIF™ staining on COMET™

CD33 was detected in immersion fixed paraffin-embedded sections of human thymus using Mouse Anti-Human CD33, Monoclonal Antibody (Catalog # MAB11371) at 15ug/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™ 647 Goat anti-Mouse IgG Secondary Antibody at 1:200 at 37 ° Celsius for 2 minutes. (Yellow; Lunaphore Catalog # DR647MS) and counterstained with DAPI (blue; Lunaphore Catalog # DR100). Specific staining was localized to the membrane. Protocol available in COMET™ Panel Builder.

CD33 in Human Hodgkin Lymphoma via seqIF™ staining on COMET™

CD33 was detected in immersion fixed paraffin-embedded sections of human Hodgkin Lymphoma using Mouse Anti-Human CD33, Monoclonal Antibody (Catalog # MAB11371) at 15ug/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™ 647 Goat anti-Mouse IgG Secondary Antibody at 1:200 at 37 ° Celsius for 2 minutes. (Yellow; Lunaphore Catalog # DR647MS) and counterstained with DAPI (blue; Lunaphore Catalog # DR100). Specific staining was localized to the membrane. Protocol available in COMET™ Panel Builder.

CD33 in Human Lymph Node via seqIF™ staining on COMET™

CD33 was detected in immersion fixed paraffin-embedded sections of human lymph node using Mouse Anti-Human CD33, Monoclonal Antibody (Catalog # MAB11371) at 15ug/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™ 647 Goat anti-Mouse IgG Secondary Antibody at 1:200 at 37 ° Celsius for 2 minutes. (Yellow; Lunaphore Catalog # DR647MS) and counterstained with DAPI (blue; Lunaphore Catalog # DR100). Specific staining was localized to the membrane. Protocol available in COMET™ Panel Builder.

Siglec‑3/CD33 in Human Tonsil.

Siglec-3/CD33 was detected in immersion fixed paraffin-embedded sections of human tonsil using Mouse Anti-Human Siglec-3/CD33 Monoclonal Antibody (Catalog # MAB11371) 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 Antigen Retrieval Reagent-Basic (Catalog # CTS013). 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.

Detection of Human Siglec‑3/CD33 by Western Blot.

Western blot shows lysates of U937 human histiocytic lymphoma cell line. PVDF membrane was probed with 2 µg/mL of Mouse Anti-Human Siglec-3/CD33 Monoclonal Antibody (Catalog # MAB11371) followed by HRP-conjugated Anti-Mouse IgG Secondary Antibody (Catalog # HAF018). A specific band was detected for Siglec-3/CD33 at approximately 55 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.

Siglec‑3/CD33 in U937 and MOLT-4 Human Cell Lines.

Siglec-3/CD33 was detected in immersion fixed U937 human histiocytic lymphoma cell line (left panel; positive stain) and MOLT-4 human acute lymphoblastic leukemia cell line (right panel; negative stain) using Mouse Anti-Human Siglec-3/CD33 Monoclonal Antibody (Catalog # MAB11371) 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 cell surfaces. View our protocol for Fluorescent ICC Staining of Cells on Coverslips.

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Applications for Human Siglec-3/CD33 Antibody

Application

Recommended Usage

COMET

15 µg/mL
Sample: Immersion fixed paraffin-embedded sections of human tonsil, thymus, Hodgkin Lymphoma and lymph node

Immunocytochemistry

8-25 µg/mL
Sample: Immersion fixed U937 human histiocytic lymphoma cell line

Immunohistochemistry

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

Western Blot

2 µg/mL
Sample: U937 human histiocytic lymphoma cell line

Reviewed Applications

Read 4 reviews rated 4.8 using MAB11371 in the following applications:

Immunocytochemistry/Immunofluorescence (1 Review)
Immunohistochemistry (2 Reviews)
Immunohistochemistry-Paraffin (1 Review)

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. See Certificate of Analysis for details.
*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: Siglec-3/CD33

Siglecs (sialic acid binding Ig-like lectins) are I-type (Ig-type) lectins belonging to the Ig superfamily. They are characterized by an N‑terminal Ig-like V-type domain which mediates sialic acid binding, followed by varying numbers of Ig-like C2-type domains (1, 2). Eleven human Siglecs have been cloned and characterized. They are sialoadhesin/CD169/Siglec-1, CD22/Siglec-2, CD33/Siglec-3, Myelin-Associated Glycoprotein (MAG/Siglec-4a) and Siglecs 5 to 11 (1‑3). To date, no Siglec has been shown to recognized any cell surface ligand other than sialic acids, suggesting that interactions with glycans containing this carbohydrate are important in mediating the biological functions of Siglecs. Siglecs 5 to 11 share a high degree of sequence similarity with CD33/Siglec-3 both in their extracellular and intracellular regions. They are collectively referred to as CD33-related Siglecs. One remarkable feature of the CD33-related Siglecs is their differential expression pattern within the hematopoietic system (1, 2). This fact, together with the presence of two conserved immunoreceptor tyrosine-based inhibition motifs (ITIMs) in their cytoplasma tails, suggests that CD33-related Siglecs are involved in the regulation of cellular activation within the immune system.

Human Siglec-3 is alternatively known as myeloid cell surface antigen CD33 and GP67. Human Siglec-3 cDNA encodes a 364 amino acid (aa) polypeptide with a hydrophobic signal peptide, an N-terminal Ig-like V-type domain, one Ig-like C2-type domains, a transmembrane region and a cytoplasmic tail (1, 4). Siglec-3 expression is restricted to cells of myelomonocytic lineage (2). It binds sialic acid preferring alpha2,3- linkage over alpha2,6- linkage (5). Studies indicated that Siglec-3 recruits SHP-1 and SHP-2 to its ITIMs (6, 7). When co-crosslinking with Fc gammaR1, Siglec-3 inhibits tyrosine phosphorylation and calcium mobilization, suggesting Siglec-3 can mediate inhibitory signals (7).

References

Crocker, P.R. and A. Varki (2001) Trends Immunol. 22:337.
Crocker, P.R. and A. Varki (2001) Immunology 103:137.
Angata, T. et al. (2002) J. Biol. Chem. 277:24466.
Simmons, D. and B. Seed (1988) J. Immunol. 141:2797.
Freeman, S.D. et al. (1995) Blood 85:2002.
Taylor, V.C. et al. (1999) J. Biol. Chem. 274:11505.
Ulyanova, T. et al. (1999) Eur. J. Immunol. 29:3440.

Long Name

Sialic Acid Binding Ig-like Lectin 3

Alternate Names

CD33, gp67, Siglec3

Entrez Gene IDs

945 (Human); 12489 (Mouse); 102117580 (Cynomolgus Monkey)

Gene Symbol

CD33

UniProt

P20138

Additional Siglec-3/CD33 Products

Product Documents for Human Siglec-3/CD33 Antibody

Product Datasheets

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COA

SDS

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Product Specific Notices for Human Siglec-3/CD33 Antibody

For research use only

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