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IL33 / Interleukin-33 · IHC design guide
Design Immunohistochemistry for IL33
Plan IL33 paraffin-section IHC around selective nuclear staining in endothelial, immune and epithelial cells (HPA tissue IHC). Use bronchial respiratory epithelium as a positive reference and adrenal glandular cells as a negative reference (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
IL33 Immunohistochemistry Experimental Design Guide
Expected localisation, validated protocols, controls and antibodies — the at-a-glance facts below, then the full design guide.
Must know before staining
| Expected localisation | Nuclear staining in select cells (HPA tissue IHC) | |
| Staining pattern | Nuclear endothelial staining; immune and epithelial subsets (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6 HIER, heat-mediated (datasheet A00113) | |
| Positive control | Bronchus+4 more · see all | |
| Negative control | Adrenal gland+4 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent across samples (standard IHC practice; not target-specific); validate locally. | |
| Caveat | Secreted IL33 may differ from the nuclear tissue pattern (UniProt) | |
| Regulation | High in high endothelial venules (UniProt) | |
| Isoform / epitope | 4 isoforms; cleavage can remove N-terminal epitopes (UniProt) |
Section 1
Recommended IL33 IHC & IF Protocols
The catalog antibody uses citrate pH 6 heat retrieval (datasheet: A00113). The four published IL33 IHC protocols below provide additional reported conditions (PMC13285577; PMC11794308; PMC10749886; PMC7060130).
| Sample | Paraffin-embedded human tonsil tissue; fixative not specified (datasheet A00113) |
| Fixation | Image fixative and duration unreported (datasheet A00113); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: Citrate pH 6, 20 min (datasheet A00113) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A00113) |
| Primary antibody | Rabbit anti-IL33, 1:100–1:500 starting range (standard) |
| Primary incubation | Overnight at 4 °C (standard) |
| Detection | Streptavidin-biotin complex (SABC), DAB chromogen (datasheet A00113) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | IL33-positive staining in respiratory epithelial cells of bronchus (HPA tissue IHC: High). HPA tissue profile: High selective nuclear expression in endothelial cells, in a subset of immune cells in non-germinal center and in a subset of cells in epithelia. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6 heat retrieval for the catalog antibody (datasheet: A00113); use each paper’s stated retrieval conditions when following its published protocol.
Section 2
What Is the Expected IL33 Staining Pattern?
Expect nuclear IL33 in endothelial and select epithelial and immune cells (HPA: Supported IHC; UniProt: no TM segment).
| Strong epithelial nuclear stain in bronchus (HPA: High). | Expected positive pattern (HPA: High in bronchial respiratory epithelium). |
| Nuclear stain in endothelial cells (HPA: tissue IHC profile). | Consistent with selective endothelial expression (HPA: tissue IHC profile). |
| Membrane-only staining (UniProt: no TM segment). | Unexpected for IL33; assess nonspecific staining (UniProt: no TM segment; general IHC practice). |
| Stain in adrenal glandular cells (HPA: Not detected). | Check cross-reactivity or endogenous detection activity (HPA: Not detected; general IHC practice). |
| Diffuse stain across the section (general IHC practice). | Suggests background; compare a no-primary control (general IHC practice). |
💡Expected IL33 appearanceCall positive when epithelial or endothelial nuclei stain; bronchial epithelium can be strong, while membrane-only stain is suspect (HPA: tissue IHC profile; UniProt: no TM segment).
| IHC antibody validation (HPA: HPA024426 and CAB007057 Supported). | These antibodies have Supported IHC status (HPA: antibody validation). |
| Processing (UniProt: full-length and truncated IL33 chains). | Epitope location determines which chains an antibody can detect (UniProt: processing; general IHC practice). |
| Secretion (UniProt: extracellular release). | Secreted IL33 may extend beyond its producing cell (UniProt: secretion). |
| IF localisation (HPA: supported nucleoplasm and vesicles). | Vesicular signal can occur; assess it separately from the expected nuclear IHC pattern (HPA: subcellular). |
| Situation | Likely cause | Next action |
|---|---|---|
| No stain in bronchial epithelium (HPA: High). | Possible assay failure (general IHC practice). | Check IHC antibody validation and optimise retrieval with controls (HPA: antibodies; general IHC practice). |
| Only membrane outlines stain (UniProt: no TM segment). | Possible nonspecific staining (general IHC practice). | Compare no-primary and known-positive controls (general IHC practice). |
| Adrenal glandular cells stain (HPA: Not detected). | Possible cross-reactivity or endogenous activity (general IHC practice). | Check no-primary and detection controls (general IHC practice). |
| Diffuse slide-wide colour (general IHC practice). | Possible background from detection or insufficient blocking (general IHC practice). | Check no-primary control; optimise blocking and washes (general IHC practice). |
| Weak nuclear stain in a positive region (HPA: bronchus High). | Retrieval or antibody dilution may need optimisation (general IHC practice). | Titrate retrieval and dilution using a positive control (general IHC practice). |
| IF shows vesicles alongside nuclei (HPA: subcellular). | Both locations are supported in ICC-IF (HPA: subcellular). | Score nuclear and vesicular signal separately (HPA: subcellular; general IF practice). |
Experiment evidence
IHC & IF images showing IL33 staining
A00113 · IHCIHC analysis of IL33 using anti-IL33 antibody (A00113). IL33 was detected in paraffin-embedded section of human tonsil . Heat mediated antig
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000137033-IL33/tissue/cerebral+cortex#imid_2091224
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000137033-IL33/tissue/colon#imid_2091142
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000137033-IL33/tissue/liver#imid_2091159
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000137033-IL33/tissue/kidney#imid_2091144
![HPA ICC-IF BJ [Human fibroblast] — IL33 (HPA022899)](https://images.proteinatlas.org/22899/1936_B1_1_blue_red_green.jpg)
HPA · ICC-IF BJ [Human fibroblast]Human Protein Atlas immunofluorescence, BJ [Human fibroblast], antibody HPA022899; green = IL33, blue = nucleus, red = microtubules.
![HPA ICC-IF BJ [Human fibroblast] — IL33 (HPA022899)](https://images.proteinatlas.org/22899/1936_B1_3_blue_red_green.jpg)
HPA · ICC-IF BJ [Human fibroblast]Human Protein Atlas immunofluorescence, BJ [Human fibroblast], antibody HPA022899; green = IL33, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for IL33 IHC & IF
🧪Run bronchus first and assess its respiratory epithelial cells for IL33 staining (HPA: High in bronchus respiratory epithelial cells). Use adrenal gland glandular cells as a negative comparison (HPA: Not detected); on the bronchus slide, cells without specific staining should show only background signal, but their identity should not be assumed from the HPA row.
Positive control tissue: Bronchus (Respiratory epithelial cells, HPA High)
Negative control tissue: Adrenal gland (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show IL33 in BJ [Human fibroblast], EFO-21, U2OS, with annotated localisation: Nucleoplasm (supported) (HPA subcellular).
Technical controls: Include a no-primary (secondary-only) control, a clonality-appropriate rabbit IgG isotype control (selected-SKU caption: rabbit primary antibody), and IL33-knockout tissue or a validated peptide-block control as a biological specificity check. For chromogenic bronchus IHC, quench endogenous peroxidase and assess endogenous biotin background because the selected-SKU detection uses a biotinylated secondary, streptavidin–biotin complex and DAB (selected-SKU caption).
⚠️Feasibility: The selected-SKU paraffin-section caption does not report a fixative, and the supplied evidence reports no IL33-specific fixation window or fixation effect (selected-SKU caption: fixative unreported). Its demonstrated IHC approach uses heat-mediated citrate retrieval at pH 6 for 20 minutes, so retrieval is a practical starting point (selected-SKU caption). The supplied evidence does not establish that frozen sections or IF/ICC are easier than paraffin IHC; bronchus sections should be checked for endogenous peroxidase or biotin background under the stated chromogenic detection method (selected-SKU caption: biotin-based DAB detection).
HPA tissue IHC evidence for IL33
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Supported — High consistency between antibody staining and RNA expression data. At least one protein variant secreted, tissue location of RNA and protein might differ and correlation is complex. Antibody staining in cells/structures not annotated, view images.). Rows are taken directly from the HPA tissue chart — click any row's HPA link to view the source.
Positive expression · recommended positive controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Bronchus | Respiratory epithelial cells | High | Protein (IHC) | HPA → |
| Cervix | Glandular cells | High | Protein (IHC) | HPA → |
| Endometrium | Ciliated epithelial cells | High | Protein (IHC) | HPA → |
| Nasopharynx | Respiratory epithelial cells | High | Protein (IHC) | HPA → |
| Stomach | Glandular cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adrenal gland | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Duodenum | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Epididymis | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Fallopian tube | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Gallbladder | Glandular cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced IL33 IHC Tips
Troubleshoot IL33 staining in paraffin sections by checking retrieval, nuclear localisation, cell identity and controls before comparing staining scores (HPA tissue IHC; UniProt O95760).
What retrieval should I try first when IL33 staining is weak in paraffin sections?
Use heat-mediated retrieval in citrate buffer at pH 6 for 20 minutes before chromogenic IHC (A00113 tissue-IHC caption). The catalog antibody stained a paraffin-embedded human tonsil section after that treatment, with 1 µg/mL primary antibody and DAB detection (A00113 tissue-IHC caption). Compare a known positive section across runs, and keep heating, cooling and primary incubation consistent when judging a change in signal (standard IHC practice). If staining remains weak, optimise retrieval time on adjacent sections while checking whether stronger heating also raises background or damages tissue morphology (standard IHC practice).
Could fixation explain weak or uneven IL33 staining?
The selected paraffin-section caption does not state a fixative, so IL33 sensitivity to any specific fixation condition is unknown for this antibody (A00113 tissue-IHC caption). Record the specimen’s actual fixative, fixation duration and processing history before comparing sections, since these variables can alter antigen accessibility in routine IHC (standard IHC practice). Run adjacent sections from the same block through the same citrate pH 6 retrieval and staining batch to isolate handling differences (A00113 tissue-IHC caption; standard IHC practice). If staining varies between specimens, compare morphology and processing records before attributing the difference to IL33 expression (standard IHC practice).
Where should convincing IL33 staining appear in tissue sections?
Prioritise cell-associated nuclear staining: tissue IHC shows high selective nuclear expression in endothelial cells and subsets of immune and epithelial cells (HPA tissue IHC). Nucleoplasm is the supported main subcellular location, with vesicles as an additional location; UniProt also lists cytoplasmic and secreted IL33 (HPA subcellular; UniProt O95760 subcellular). Assess the stained structure against a counterstain and tissue architecture before assigning it to a cell type (standard IHC practice). Diffuse extracellular colour alone cannot establish the source cell, because at least one IL33 protein variant is secreted and RNA and protein locations may differ (HPA tissue IHC).
How might IL33 processing change what this antibody detects?
IL33 has 4 annotated isoforms and listed chains spanning residues 1–270, 95–270, 99–270 and 109–270 (UniProt O95760 processing and isoforms). A stain cannot distinguish those forms unless the antibody’s recognised epitope and its presence in each form are established (standard IHC interpretation). Check the catalog antibody’s epitope information before claiming that a nuclear or extracellular signal represents a particular processed form (standard IHC interpretation; UniProt O95760 subcellular). UniProt lists no transmembrane segment, glycosylation sites or modified residues, but those annotations alone do not establish epitope exposure after tissue processing (UniProt O95760 topology and PTMs; standard IHC interpretation).
How can IF help verify the cells and compartment stained by IL33 IHC?
Use IF as a separate localisation check, multiplexing IL33 with a validated marker for the suspected endothelial, immune or epithelial cell population (HPA tissue IHC; standard IF practice). Choose fluorophores and imaging settings after checking autofluorescence in an unstained section, so tissue background does not masquerade as IL33 signal (standard IF practice). If testing an intracellular epitope, optimise permeabilisation to give antibody access to the nucleoplasm or vesicles while preserving morphology (HPA subcellular; standard IF practice). Compare signal with a nuclear counterstain, and do not transfer the catalog antibody’s paraffin-section retrieval conditions to IF without validation (A00113 tissue-IHC caption; standard IF practice).
How do I reduce diffuse or vascular background without losing IL33 signal?
Check a no-primary control and inspect the distribution of DAB colour before changing antibody concentration; endogenous enzyme activity and nonspecific binding can mimic weak staining (standard IHC practice). The selected tonsil example used 10% goat serum before 1 µg/mL primary antibody and a biotin-based DAB detection system (A00113 tissue-IHC caption). Apply an appropriate endogenous peroxidase block and, with biotin-based detection, assess endogenous biotin as possible sources of background (standard chromogenic IHC practice). Shorten or dilute one staining step at a time, then confirm that expected cell-associated nuclear signal remains visible (HPA tissue IHC; standard IHC practice).
What should I score when comparing IL33 across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring, because HPA reports selective nuclear staining in endothelial cells and subsets of immune and epithelial cells (HPA tissue IHC). For that population, report the percentage of positive nuclei and an intensity-weighted H-score, using consistent thresholds and counterstain review across specimens (standard IHC quantification). If distribution matters, report positive-cell density per mm² of evaluable tissue or the defined compartment (standard IHC quantification). Normalise counts to all evaluable cells of the same type or to measured tissue area, and exclude folds, necrosis and section edges consistently (standard IHC quantification).
How can I distinguish genuine IL33 staining from section artefacts?
A convincing result follows recognisable cells and favours nuclei in the endothelial, immune or epithelial subsets described for tissue IHC (HPA tissue IHC). Check whether staining instead tracks section edges, folds or necrotic areas, and compare suspicious DAB deposits with a no-primary control and peroxidase block (standard IHC practice). Cytoplasmic, vesicular or extracellular signal is biologically possible, but its location alone cannot identify a producing cell or a specific processed form (UniProt O95760 subcellular and processing; HPA subcellular). Resolve uncertain patterns with cell-type markers, morphology and independently validated controls before interpreting differences between samples as altered IL33 expression (standard IHC practice).
Boster reagents
Best IL33 / Interleukin-33 IHC Antibodies
A00113 has real IHC data from human tonsil paraffin sections (A00113 image caption); the catalog supplies no IF figure or IF validation (catalog).
Real IHC data
Anti-IL33 Antibody ®
Cat # A00113
A00113 is listed for human IHC (catalog: applications and reactivity). Its IHC figure shows staining in a human tonsil paraffin section (A00113 image caption).
Which to pick: Choose A00113 for human tissue IHC in paraffin sections; its caption reports citrate retrieval at pH 6 for 20 minutes, and does not report the fixative (A00113 image caption). There is no catalog supported IF/ICC or cross species choice here: A00113 lists human reactivity and IHC, but no IF/ICC application or other species (catalog: applications and reactivity).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry O95760 (IL33_HUMAN, Interleukin-33).
- Human Protein Atlas. IL33 tissue IHC expression (reliability: Supported).
- Human Protein Atlas. IL33 subcellular location (ICC-IF): Mainly localized to the nucleoplasm. In addition localized to vesicles..
- Human Protein Atlas. IL33 antibody validation summary (3 antibodies).
- Trophoblast aging driven by IL33 deficiency elevates recurrent pregnancy loss risk through SNAP29 lactylation-mediated autophagy impairment. Autophagy 2026 — PMC13285577.
- Efferocytosis-related gene IL33 predicts prognosis and immune response and mediates proliferation and migration in vitro and in vivo of breast cancer. Frontiers in pharmacology 2025 — PMC11794308.
- Effect of three clinical therapies on cytokines modulation in the hip articular cartilage and bone improvement in rat early osteonecrosis of the femoral head. Biomedical journal 2023 — PMC10749886.
- ILC2s amplify PD-1 blockade by activating tissue-specific cancer immunity. Nature 2020 — PMC7060130.
- PubMed PMID:12819012 — UniProt-cited evidence.
- PubMed PMID:10566975 — UniProt-cited evidence.
- PubMed PMID:16286016 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for IL33 (Interleukin-33). Expected localisation Nuclear staining in select cells (HPA tissue IHC). Antigen retrieval: Citrate pH 6 HIER, heat-mediated (datasheet A00113). Validated IHC antibody: A00113 at 1:100–1:500 (standard starting range) (the product's own IHC validation image is shown). Controls (HPA): positive Bronchus (Respiratory epithelial cells); negative Adrenal gland. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human tonsil tissue; fixative not specified (datasheet A00113); 2. Fixation: Image fixative and duration unreported (datasheet A00113); verify before use.; 3. Sectioning: 4–5 µm sections on charged slides (standard); 4. Deparaffinisation: Xylene, graded ethanol series to water (standard); 5. Antigen retrieval: Heat retrieval: Citrate pH 6 , 20 min (datasheet A00113); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A00113); 8. Primary antibody: Rabbit anti-IL33, 1:100–1:500 starting range (standard); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across samples (standard IHC practice; not target-specific); validate locally.; Caveat: Secreted IL33 may differ from the nuclear tissue pattern (UniProt); Isoform / epitope: 4 isoforms; cleavage can remove N-terminal epitopes (UniProt); Regulation: High in high endothelial venules (UniProt). Evidence: UniProt O95760, HPA tissue IHC · Supported, HPA ICC-IF · Mainly localized to the nucleoplasm. In addition localized to…, Datasheet A00113, PMC13285577, PMC11794308. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/IL33/immunohistochemistry-ihc-guide.