|
How to Cite 1. For In-Text Citation (Materials & Methods): 2. For Key Resources Table: |
||
|
Toll Free: (877) 796-6397 -- USA and Canada only -- |
Fax: +1-832-582-8590 Orders: +1-832-582-8158 |
Tech Support: +1-832-582-8158 Ext:3 Please provide your Order Number in the email. We strive to reply to |
Biological Description
| Specificity | IL-18 Rα/IL-1 R5 Antibody (Mouse mAb) [F14H3] detects endogenous levels of total IL-18 Rα/IL-1 R5 protein. |
|---|---|
| Background | IL‑18 Rα (also known as IL‑18R1, IL‑1 R5, or IL‑1Rrp) is the ligand‑binding chain of the heterodimeric IL‑18 receptor complex within the IL‑1 receptor/Toll‑like receptor superfamily, forming with IL‑18Rβ (IL‑18RAP/IL‑1 R7) the high‑affinity signaling receptor that couples the proinflammatory cytokine IL‑18 to downstream MyD88‑dependent pathways in hematopoietic and non‑hematopoietic cells. The IL‑18 Rα polypeptide is a type I transmembrane receptor with a signal peptide, three extracellular Ig‑like domains that contact IL‑18, a single transmembrane segment, and a cytoplasmic Toll/IL‑1 receptor (TIR) domain that nucleates signaling complexes; IL‑18 first docks onto IL‑18 Rα with low affinity, then recruits IL‑18Rβ to assemble a heterotrimeric receptor–cytokine complex that supports high‑affinity binding and signal initiation at the plasma membrane. Engagement of IL‑18 Rα by mature IL‑18 positions the TIR domain to recruit the adaptor MyD88, followed by association of IL‑1R‑associated kinases (IRAKs) and TRAF6, which activate NF‑κB‑inducing kinase and the IκB kinase complex, leading to phosphorylation and proteasomal degradation of IκB and nuclear translocation of NF‑κB, while parallel TRAF‑dependent branches stimulate MAPKs including p38 and MEK–ERK, as well as PI3K/Akt, so that IL‑18Rα‑containing complexes drive coordinated transcriptional programs for IFN‑γ production, chemokine induction (such as MCP‑1), and broader inflammatory gene expression. This signaling module functions in multiple immune contexts: IL‑18 Rα is expressed on T helper subsets, NK cells, B cells, monocytes, and other hematopoietic lineages, where its activation enhances Th1 polarization, boosts cytotoxic effector functions, and synergizes with IL‑12 and related cytokines to amplify IFN‑γ output; receptor expression on additional cell types, including vascular and intestinal cells, extends IL‑18 responsiveness to barrier and stromal compartments and links epithelial or endothelial stress to innate and adaptive immune activation. The receptor chain is subject to regulatory modulation at several levels: inducible expression of IL‑18 Rα on lymphoid cells defines IL‑18‑responsive subsets; decoy or truncated receptor isoforms lacking the TIR domain and soluble antagonists such as IL‑18 binding protein compete with IL‑18 Rα for ligand and dampen signaling; and related cytokines like IL‑37 can form complexes with IL‑18 Rα and inhibitory coreceptors, shifting the balance toward anti‑inflammatory outputs, so the abundance and context of IL‑18 Rα critically shape how IL‑18 signaling is interpreted in tissues. Pathophysiologically, IL‑18 Rα participates in chronic inflammatory and autoimmune diseases, where increased IL‑18–IL‑18R signaling contributes to vascular inflammation, atherosclerotic lesion development, and tissue damage, as well as in lymphoid malignancies and B‑cell lymphoproliferative disorders that retain or upregulate IL‑18R expression and can respond to IL‑18 with altered growth or cytokine profiles, indicating a role for IL‑18 Rα–containing complexes in both host defense and disease‑associated immune dysregulation. |
Usage Information
| Application | IHC, IF, FCM | Dilution |
|
||||||
|---|---|---|---|---|---|---|---|---|---|
| Reactivity | Human | ||||||||
| Source | Mouse Monoclonal Antibody | MW | 62 kDa | ||||||
| Storage Buffer | PBS, pH 7.2+50% Glycerol+0.05% BSA+0.01% NaN3 | Storage (from the date of receipt) |
-20°C (avoid freeze-thaw cycles), 2 years | ||||||
| IHC |
Experimental Protocol：
Deparaffinization/Rehydration
1. Deparaffinize/hydrate sections:
2. Incubate sections in three washes of xylene for 5 min each.
3. Incubate sections in two washes of 100% ethanol for 10 min each.
4. Incubate sections in two washes of 95% ethanol for 10 min each.
5. Wash sections two times in dH2O for 5 min each.
6.Antigen retrieval: For Citrate: Heat slides in a microwave submersed in 1X citrate unmasking solution until boiling is initiated; continue with 10 min at a sub-boiling temperature (95°-98°C). Cool slides on bench top for 30 min.
Staining
1. Wash sections in dH2O three times for 5 min each.
2. Incubate sections in 3% hydrogen peroxide for 10 min.
3. Wash sections in dH2O two times for 5 min each.
4. Wash sections in wash buffer for 5 min.
5. Block each section with 100–400 µl of blocking solution for 1 hr at room temperature.
6. Remove blocking solution and add 100–400 µl primary antibody diluent in to each section. Incubate overnight at 4°C.
7. Remove antibody solution and wash sections with wash buffer three times for 5 min each.
8. Cover section with 1–3 drops HRPas needed. Incubate in a humidified chamber for 30 min at room temperature.
9. Wash sections three times with wash buffer for 5 min each.
10. Add DAB Chromogen Concentrate to DAB Diluent and mix well before use.
11. Apply 100–400 µl DAB to each section and monitor closely. 1–10 min generally provides an acceptable staining intensity.
12. Immerse slides in dH2O.
13. If desired, counterstain sections with hematoxylin.
14. Wash sections in dH2O two times for 5 min each.
15. Dehydrate sections: Incubate sections in 95% ethanol two times for 10 sec each; Repeat in 100% ethanol, incubating sections two times for 10 sec each; Repeat in xylene, incubating sections two times for 10 sec each.
16. Mount sections with coverslips and mounting medium.
|
| IF |
Experimental Protocol:
Sample Preparation
1. Adherent Cells: Place a clean, sterile coverslip in a culture dish. Once the cells grow to near confluence as a monolayer, remove the coverslip for further use.
2. Suspension Cells: Seed the cells onto a clean, sterile slide coated with poly-L-lysine.
3. Frozen Sections: Allow the slide to thaw at room temperature. Wash it with pure water or PBS for 2 times, 3 minutes each time.
4. Paraffin Sections: Deparaffinization and rehydration. Wash the slide with pure water or PBS for 3 times, 3 minutes each time. Then perform antigen retrieval.
Fixation
1. Fix the cell coverslips/spots or tissue sections at room temperature using a fixative such as 4% paraformaldehyde (4% PFA) for 10-15 minutes.
2. Wash the sample with PBS for 3 times, 3 minutes each time.
Permeabilization
1.Add a detergent such as 0.1–0.3% Triton X-100 to the sample and incubate at room temperature for 10–20 minutes.
(Note: This step is only required for intracellular antigens. For antigens expressed on the cell membrane, this step is unnecessary.)
Wash the sample with PBS for 3 times, 3 minutes each time.
Blocking
Add blocking solution and incubate at room temperature for at least 1 hour. (Common blocking solutions include: serum from the same source as the secondary antibody, BSA, or goat serum.)
Note: Ensure the sample remains moist during and after the blocking step to prevent drying, which can lead to high background.
Immunofluorescence Staining (Day 1)
1. Remove the blocking solution and add the diluted primary antibody.
2. Incubate the sample in a humidified chamber at 4°C overnight.
Immunofluorescence Staining (Day 2)
1. Remove the primary antibody and wash with PBST for 3 times, 5 minutes each time.
2. Add the diluted fluorescent secondary antibody and incubate in the dark at 4°C for 1–2 hours.
3. Remove the secondary antibody and wash with PBST for 3 times, 5 minutes each time.
4. Add diluted DAPI and incubate at room temperature in the dark for 5–10 minutes.
5. Wash with PBST for 3 times, 5 minutes each time.
Mounting
1. Mount the sample with an anti-fade mounting medium.
2. Allow the slide to dry at room temperature overnight in the dark.
3. Store the slide in a slide storage box at 4°C, protected from light.
|
References
|