Immunohistochemistry, Western Blot, ELISA Capture (Matched Antibody Pair), Neutralization, Intracellular Staining by Flow Cytometry, Immunocytochemistry, CyTOF-ready

Cited:

Immunohistochemistry, Immunohistochemistry-Paraffin, Immunohistochemistry-Frozen, Western Blot, Neutralization, Flow Cytometry, Immunofluorescence, Immunocytochemistry, Immunoprecipitation, Chromatin Immunoprecipitation (ChIP), ELISA Development, ELISA Development (Capture), ELISpot Development, In vivo assay, Neutralization Control

Label

Unconjugated

Antibody Source

Polyclonal Goat IgG

View all IL-33 Antibodies »

Product Specifications

Immunogen

E. coli-derived recombinant mouse IL-33
Ser109-Ile266
Accession # Q8BVZ5

Specificity

Detects mouse IL-33 in ELISAs and Western blots. In sandwich immunoassays, less than 2% cross-reactivity with recombinant human IL‑33 is observed.

Clonality

Polyclonal

Host

Goat

Isotype

IgG

Endotoxin Level

<0.10 EU per 1 μg of the antibody by the LAL method.

Scientific Data Images for Mouse IL-33 Antibody

Detection of Mouse IL‑33 by Western Blot.

Western blot shows lysates of HEK293T human embryonic kidney cell line either mock transfected or transfected with full length mouse IL-33. PVDF membrane was probed with 0.4 µg/mL of Goat Anti-Mouse IL-33 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF3626) followed by HRP-conjugated Anti-Goat IgG Secondary Antibody (Catalog # HAF109). A specific band was detected for IL-33 at approximately 37 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.

IL‑33 in Mouse Spleen.

IL-33 was detected in immersion fixed frozen sections of mouse spleen using Goat Anti-Mouse IL-33 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF3626) at 5 µg/mL overnight at 4 °C. Tissue was stained using the Anti-Goat HRP-DAB Cell & Tissue Staining Kit (brown; Catalog # CTS008) and counterstained with hematoxylin (blue). Specific staining was localized to cytoplasm in lymphocytes. View our protocol for Chromogenic IHC Staining of Frozen Tissue Sections.

IL‑33 in bEnd.3 Mouse Cell Line.

IL-33 was detected in immersion fixed bEnd.3 mouse endothelioma cell line using Goat Anti-Mouse IL-33 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF3626) at 10 µg/mL for 3 hours at room temperature. Cells were stained using the NorthernLights™ 557-conjugated Anti-Goat IgG Secondary Antibody (red, upper panel; Catalog # NL001) and counterstained with DAPI (blue, lower panel). View our protocol for Fluorescent ICC Staining of Cells on Coverslips.

Detection of IL-33 in bEND.3 cells by Flow Cytometry

bEND.3 cells were stained with Goat Anti-Mouse IL-33 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF3626, filled histogram) or isotype control antibody (Catalog # AB-108-C, open histogram) followed by Phycoerythrin-conjugated Anti-Goat IgG Secondary Antibody (Catalog # F0107). To facilitate intracellular staining, cells were fixed with Flow Cytometry Fixation Buffer (Catalog # FC004) and permeabilized with Flow Cytometry Permeabilization/Wash Buffer I (Catalog # FC005). View our protocol for Staining Intracellular Molecules.

Detection of IL‑33 in Mouse Splenocytes by Flow Cytometry.

Mouse splenocytes were treated for 5 hours with 50 ng/mL PMA and 1 µg/mL Ca²⁺ionomycin then stained with Goat Anti-Mouse IL-33 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF3626, filled histogram) or isotype control antibody (Catalog # AB-108-C, open histogram), followed by Phycoerythrin-conjugated Anti-Goat IgG Secondary Antibody (Catalog # F0107). To facilitate intracellular staining, cells were fixed with paraformaldehyde and permeabilized with saponin.

Proliferation Induced by IL‑33 and Neutralization by Human IL‑33 Antibody.

Recombinant Mouse IL-33 (Catalog # 3626-ML) stimulates proliferation in the D10.G4.1 mouse helper T cell line in a dose-dependent manner (orange line), as measured by Resazurin (Catalog # AR002). Proliferation elicited by Recombinant Mouse IL-33 (0.25 ng/mL) is neutralized (green line) by increasing concentrations of Goat Anti-Mouse IL-33 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF3626). The ND₅₀ is typically 10-50 ng/mL.

Detection of Mouse IL-33 by Immunohistochemistry

IL-33/ST2 axis in tendon healing in vivo.Kinetics of (a) Il33 and (b) soluble St2 gene expression post injury. Data are mean fold change±s.d. showing relative expression to 18s housekeeping gene (pooled data from four mice per group performed on four sequential occasions, n=16 per condition). *P<0.05, **P<0.01 versus control (WT) mice (ANOVA) +P<0.05,++P<0.01, NS not significant; control (WT) injured versus St2−/− injured. (c) Immunohistochemistry showing IL-33 and ST2 expression in tendon biopsies of WT and St2−/− mice day 1 post injury. IgG control shown in bottom left of pictures. Black horizontal line indicates 50 μm. (d) Col3 mRNA and (e) Collagen 3 protein levels in tendon biopsies of WT and St2−/− mice post injury. (f) Col1 mRNA and (g) Collagen 1 protein levels in tendon biopsies of WT and St2−/− mice post injury. For (d–g) data are mean±s.d. of duplicate samples, representative of four mice per condition (n=16). *P<0.05, **P<0.01 versus control (WT) mice. +P<0.05, ++P<0.01 control (WT) injured versus St2−/− injured mice (ANOVA). mRNA graphs show relative expression to 18s housekeeping gene. (h) Percentage change in tendon strength for WT and St2−/− mice post injury. Data are mean±s.d., representative of four mice per condition (n=16). *P<0.05, **P<0.01 versus control mice. #P<0.05 St2−/− injured versus WT injured mice (Mann–Whitney U-test). Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/25857925), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Mouse IL-33 by Western Blot

HMGB1 increases IL-33 expression and acts as an autocrine factor in enhancing IL-33 expression in a partial TLR-4 dependent fashion during cyclic stretch.(A) MLE-12 cells were treated with 3 μg/ml HMGB1 or control solution before 6h cyclic stretch and IL-33 production was measured by ELISA. (B) MLE-12 cells were treated with 10 μg/ml HMGB1 neutralizing antibody (2G7) or control solution before 6h cyclic stretch and IL-33 production was detected by Western blot. beta-actin served as loading control. (C) MLE-12 cells transfecting with non-specific control siRNA or TLR-4-specific siRNA were treated with 3 μg/ml HMGB1 or control solution before stretch. IL-33 production in each group was measured with ELISA. **P<0.01, ***P<0.001 when compared between groups denoted by horizontal lines. Each stretching group collected at least from three wells and represented a single experiment, the bar graphs illustrate data representative of three independent experiments. Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/28898270), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human IL-33 by Immunohistochemistry

IL-33/ST2 expression in human tendon.(a) IL-33, (b) soluble ST2 (sST2) and (c) mST2 mRNA expression in human tendon samples. Fold change in gene expression of IL-33, sST2 and mST2 in control (semi-membranosus tendon, n=10), torn supraspinatus tendon (established pathology) and matched subscapularis human tendon samples (early pathology; n=17). Data are mean±s.d. relative to the housekeeping gene18S (mean of duplicate analysis). *P<0.05, **P<0.01, ***P<0.001 versus control (Student's t-test). (d) Immunostaining of IL-33 and ST2 in control (n=10), torn tendon (n=17) and early tendinopathy (n=17). Graphs illustrate modified Bonar scoring based on 10 high-power fields. Data are mean±s.d. *P<0.05, **P<0.01 versus control (Student's t-test). Scale bar, 65 μm. (e) Fold change in gene expression of IL33 and ST2 24 h post incubation with tumour necrosis factor (TNF-alpha), IL-1 beta or in combination depicting relative expression to media alone utilizing housekeeping gene GAPDH. Data are mean±s.d. of triplicate samples, representative of three individual patient samples. *P<0.05, **P<0.01 versus control (media) (Student's t-test). (f) IL-33 immunostaining of human tendon explants cultured for 24 h with medium (control), 100 ng ml−1 TNF alpha or 100 ng ml−1 TNF alpha+100 ng ml−1 IL-1 beta. (g) Fold change in COL1 and COL3 mRNA expression in human tendon explants cultured for 24 h with rhIL-33, relative to housekeeping gene GAPDH. (h) Time course of COL1 and COL3 mRNA expression following incubation with rhIL-33, relative to housekeeping gene GAPDH. (i) Collagen 1 and 3 protein expression in human tendon explants 24 h post incubation with rhIL-33. For (g–i) data are mean±s.d. of triplicate samples, representative of three individual patients. *P<0.05, **P<0.01 versus control (Student's t-test). Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/25857925), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Mouse Mouse IL-33 Antibody by Immunohistochemistry

Prior Nb exposure results in earlier and exacerbated HSV-2 pathology. (I and J) (I) Vaginal IL-33 measured by ELISA (day 2) and (J) IF staining of vaginal tissue (day 3; n = 4). Data are representative of two independent experiments with 4–6 mice per group (mean ± SEM). Statistical significance was calculated by two-way analysis of variance (ANOVA) with Bonferroni correction for multiple comparisons and Mann-Whitney t test. ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001, ns, not significant. Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/33857419), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of IL-33 by Immunocytochemistry/ Immunofluorescence

IL13 expression in FAPs is upregulated in Tchp−/− mice. A) qRT‐PCR analysis of RNA isolated from whole TA muscle of WT and Tchp−/− mice (n = 3 or 4) at 3 dpi. B) Immunofluorescence for IL13 (red), hematopoietic cells (CD45, green), and nuclei (Hoechst33342, blue) in TA muscle cross‐sections of WT and Tchp−/− mice at 3 dpi. Lower magnification images are shown in Figure S6A, Supporting Information. Scale bars, 10 µm. C) Quantification of the IL4‐, IL13‐, or IL33‐stained area per 40× view in (B) and Figure S6A,B (Supporting Information) (n = 6 or 7 fields each). D) Quantification of the IL13‐stained area per 40× view excluding the CD45‐stained area in (B) (n = 7 fields each). E) Immunofluorescence for IL4 (red), IL13 (red), and IL33 (red), PDGFR alpha+ (green), and nuclei (Hoechst33342, blue) in PDGFR alpha+ FAPs isolated from hindlimbs of WT and Tchp−/‐ mice. Scale bars: 20 µm. All data are the mean ± S.D from 3 to 5 mice. *p < 0.05, **p < 0.01, N.S., not significant; two‐tailed unpaired Student's t‐tests. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/36373718), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of IL-33 by Immunocytochemistry/ Immunofluorescence

Mouse IL-33 ELISA Standard Curve

Recombinant Mouse IL-33 (Catalog # 3626-ML) was serially diluted and captured by Goat Anti-Mouse IL-33 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF3626) coated on a Clear Polystyrene Microplate (Catalog # DY990). Goat Anti-Mouse IL-33 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF3626) was biotinylated and incubated with the protein captured on the plate. Detection of the standard curve was achieved by incubating Streptavidin-HRP (Catalog # DY998)

Mouse IL-33 ELISA Standard Curve

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Applications for Mouse IL-33 Antibody

Application

Recommended Usage

CyTOF-ready

Ready to be labeled using established conjugation methods. No BSA or other carrier proteins that could interfere with conjugation.

Immunocytochemistry

5-15 µg/mL
Sample: Immersion fixed bEnd.3 mouse endothelioma cell line

Immunohistochemistry

5-15 µg/mL
Sample: Immersion fixed frozen sections of mouse spleen

Intracellular Staining by Flow Cytometry

0.25 µg/10⁶ cells
Sample: bEND.3 mouse endothelioma cell line or Mouse splenocytes treated with PMA and Ca²⁺ ionomycin, fixed with paraformaldehyde, and permeabilized with saponin

Western Blot

0.4 µg/mL
Sample: HEK293T human embryonic kidney cell line transfected with full length mouse IL-33

Neutralization

Measured by its ability to neutralize IL‑33-induced proliferation in the D10.G4.1 mouse helper T cell line. Schmitz, J. et al. (2005) Immunity 23:479. The Neutralization Dose (ND₅₀) is typically 10-50 ng/mL in the presence of 0.25 ng/mL Recombinant Mouse IL‑33.

Mouse IL-33 Sandwich Immunoassay

ELISA Capture (Matched Antibody Pair)

Recommended Concentration: 0.2-0.8 µg/mL
Use in combination with these reagents:

Detection Reagent: Mouse IL-33 Biotinylated Antibody (Catalog # BAF3626)
Standard: Recombinant Mouse IL-33 Protein (Catalog # 3626-ML)

Please Note: Optimal dilutions of this antibody should be experimentally determined.

Reviewed Applications

Read 7 reviews rated 4.6 using AF3626 in the following applications:

ELISA (2 Reviews)
Immunocytochemistry (1 Review)
Immunocytochemistry/Immunofluorescence (2 Reviews)
Immunohistochemistry-Frozen (1 Review)
Western Blot (1 Review)

Formulation, Preparation, and Storage

Purification

Antigen Affinity-purified

Reconstitution

Reconstitute at 0.2 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: IL-33

IL-33, also known as NF-HEV and DVS 27, is a 30 kDa proinflammatory protein that may also regulate gene transcription (1-3). DVS 27 was identifed as a gene that is upregulated in vasospastic cerebral arteries (1). NF-HEV was described as a nuclear factor that is preferentially expressed in the endothelial cells of high endothelial venules relative to endothelial cells from other tissues (2). IL-33 was identified based on sequence and structural homology with IL-1 family cytokines (3). DVS 27, NF-HEV, and IL-33 share 100% amino acid sequence identity. IL-33 is constitutively expressed in smooth muscle and airway epithelia. It is upregulated in arterial smooth muscle, dermal fibroblasts, and keratinocytes following IL-1 alpha or IL-1 beta stimulation (1, 3). Similar to IL-1, IL-33 can be cleaved in vitro by caspase-1, generating an N-terminal fragment that is slightly shorter than the C-terminal fragment (3, 4). The N-terminal portion of full length IL-33 contains a predicted bipartite nuclear localization sequence and a homeodomain-like helix-turn-helix DNA binding domain. By immunofluorescence, full length IL-33 localizes to the nucleus in HUVECs and transfectants (2). The C-terminal fragment, corresponding to mature IL-33, binds and triggers signaling through mast cell IL-1 R4/ST2L, a longtime orphan receptor involved in the augmentation of Th2 cell responses (3, 5-7). A ternary signaling complex is formed by the subsequent association of IL-33 and ST2L with IL-1R AcP (8). Stimulation of Th2 polarized lymphocytes with mature IL-33 in vitro induces IL-5 and IL-13 secretion (3). In vivo administration of mature IL-33 promotes increased production of IL-5, IL-13, IgE, and IgA, as well as splenomegaly and inflammatory infiltration of mucosal tissues (3). Full length and mature mouse IL-33 share approximately 55% and 90% aa sequence identity with human and rat IL-33, respectively. Mouse IL-33 shares less than 25% aa sequence identity with other IL-1 family proteins.

References

Onda, H. et al. (1999) J. Cereb. Blood Flow Metab. 19:1279.
Baekkevold, E.S. et al. (2003) Am. J. Pathol. 163:69.
Schmitz, J. et al. (2005) Immunity 23:479.
Black, R.A. et al. (1989) J. Biol. Chem. 264:5323.
Xu, D. et al. (1998) J. Exp. Med. 187:787.
Lohning, M. et al. (1998) Proc. Natl. Acad. Sci. USA 95:6930.
Dinarello, C.A. (2005) Immunity 23:461.
Chackerian, A.A. et al. (2007) J. Immunol. 179:2551.

Long Name

Interleukin 33

Alternate Names

C9orf26, DVS27, IL33, NF-HEV

Entrez Gene IDs

90865 (Human); 77125 (Mouse)

Gene Symbol

IL33

UniProt

Q8BVZ5

Additional IL-33 Products

Product Documents for Mouse IL-33 Antibody

Product Datasheets

Download Product Datasheets

COA

SDS

Download SDS

Product Specific Notices for Mouse IL-33 Antibody

For research use only

Citations
Reviews

Calculators
Flow Cytometry