IL33 / Interleukin-33 · IHC design guide

Design Immunohistochemistry for IL33

Plan IL33 IHC in paraffin sections using bronchial respiratory epithelium as a high-staining reference (HPA tissue IHC). The catalog antibody’s IHC dilution is 0.5–1 µg/mL (datasheet A00113); assess nuclear staining by cell type (HPA tissue IHC).

Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use

Immunohistochemistry protocol sheet for IL33 (IHC for IL33): expected localisation Selective nuclear staining in endothelial cells (HPA tissue IHC), antibody A00113, validated IHC image, and IHC protocol steps — Printable **IL33** IHC protocol sheet — expected localisation Selective nuclear staining in endothelial cells (HPA tissue IHC), antibody A00113, controls and protocol steps. Open the full IL33 IHC guide →

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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	Selective nuclear staining in endothelial cells (HPA tissue IHC)
	Staining pattern	Nuclear staining in endothelium and subsets of immune and epithelial cells (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 sections. (standard IHC practice; not target-specific)
	Caveat	Secretion may reduce intracellular staining (UniProt)
	Regulation	Expression varies by tissue and cell type (HPA tissue IHC)
	Isoform / epitope	4 isoforms; cleavage can remove N-terminal epitopes (UniProt)

Section 1

Recommended IL33 IHC & IF Protocols

Compare the catalog antibody’s IHC-P protocol (datasheet: A00113) with published IL33 IHC protocols from PMC13285577 and PMC11794308.

Recommended immunohistochemistry (IHC-P) protocol parameters
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 heat-mediated citrate pH 6 retrieval (datasheet: A00113); the published protocols also use citrate or Tris–EDTA (PMC13285577) and pressure-cooker Tris–EDTA (PMC11794308).

Section 2

What Is the Expected IL33 Staining Pattern?

IL33 should appear mainly in nuclei, with selective staining of endothelial cells, subsets of immune cells outside germinal centers, and some epithelial cells (HPA tissue IHC: Supported). Nucleoplasmic localization is supported by ICC-IF, with additional vesicular localization (HPA subcellular). IL33 has no transmembrane segment, while UniProt also records cytoplasmic and secreted forms (UniProt O95760 topology and subcellular location).

What am I looking at on my slide?
Distinct nuclear staining in respiratory epithelium of bronchus or nasopharynx, or glandular cells of cervix or stomach.	This matches high staining in those cell types and the selective nuclear tissue profile (HPA tissue IHC). Judge the named cells and compartment together; staining elsewhere on the section does not establish the expected pattern.
Strong, continuous cell-surface staining dominates, with little nuclear signal.	A membrane-dominant pattern conflicts with the supported nuclear localization and absence of a transmembrane segment (HPA tissue IHC; UniProt O95760 topology). Investigate nonspecific staining before calling these cells IL33-positive. Limited extranuclear signal needs separate assessment because secretion is documented (UniProt O95760 subcellular location).
Many unrelated cells stain as strongly as the expected epithelial or endothelial population.	Widespread staining is less consistent with HPA's selective cell distribution (HPA tissue IHC). Consider antibody cross-reactivity or endogenous chromogenic detection activity as possible explanations (general IHC practice); compare the pattern with a detection-only control.
A uniform haze covers nuclei, cytoplasm, and tissue spaces.	Diffuse background cannot establish IL33 localization; distinguish it from bounded nuclear staining in the expected cells (HPA tissue IHC). General IHC causes include excessive antibody or detection reagent, inadequate blocking, and incomplete washing (general IHC practice).
No nuclear signal appears in a known-positive tissue, including bronchial respiratory epithelium.	This is discordant with the reported high staining in that cell population (HPA tissue IHC). Check tissue identity and assay performance before interpreting the specimen as IL33-negative; the HPA tissue profile does not establish target-specific retrieval or fixation sensitivity.

💡Expected IL33 appearanceCall a positive result when selected endothelial, immune, or epithelial cells show clear nuclear staining, with high signal possible in the listed bronchial, nasopharyngeal, cervical, and stomach cell populations (HPA tissue IHC); diffuse haze or dominant membrane staining is suspect (HPA tissue IHC; UniProt O95760 topology).

How each factor affects the staining
Cell population and tissue choice	HPA reports high staining in bronchial and nasopharyngeal respiratory epithelium and cervical and stomach glandular cells, but no detection in adrenal or duodenal glandular cells (HPA tissue IHC). Compare the same annotated cell type, not an entire tissue, when selecting controls.
Nuclear versus vesicular localization	Nucleoplasm is the supported main ICC-IF location; vesicles are an additional supported location (HPA subcellular). UniProt also lists nucleus, cytoplasm, secretory vesicles, and secretion (UniProt O95760 subcellular location). Extranuclear staining therefore needs cellular context rather than automatic rejection.
Processing and epitope location	UniProt lists full-length IL33 and processed chains beginning at residues 95, 99, or 109 (UniProt O95760 processing). Their detection may depend on the antibody epitope; no epitope is supplied here, so these records cannot predict which forms the catalog antibody recognizes.
Secretion and tissue concordance	IL33 can be released extracellularly, and HPA cautions that RNA and protein locations may differ for a secreted variant (UniProt O95760 subcellular location; HPA tissue IHC reliability description). Interpret extracellular signal alongside the nuclear cell pattern; RNA abundance alone does not identify a stained cell.
Antibody validation and isoforms	The listed tissue-IHC validation is Supported for HPA024426 and CAB007057; HPA022899 has Supported ICC validation without a listed IHC status (HPA antibodies). UniProt lists 4 isoforms (UniProt O95760 isoforms). Neither record establishes equal IHC detection of every isoform.

Why is my staining missing, weak or wrong?
Situation	Likely cause	Next action
No signal in a positive control section.	Bronchial respiratory epithelium is reported as High, so absent staining there raises an assay or specimen concern (HPA tissue IHC).	Confirm the annotated cell population and control section; review deparaffinisation, retrieval, primary antibody, and detection steps as general IHC practice. Do not infer IL33-specific fixation sensitivity from HPA.
Staining is weak or variable across the section.	HPA describes selective expression, including only subsets of immune and epithelial cells; some apparent variation can reflect cell composition (HPA tissue IHC).	Score comparable cell populations and nuclear signal within each region (HPA tissue IHC). If the positive control also varies, review section handling and staining consistency (general IHC practice).
Brown reaction product appears throughout nuclei and tissue spaces.	A section-wide deposit differs from selective nuclear IL33 staining (HPA tissue IHC); endogenous detection activity or excess background is possible (general IHC practice).	Compare a detection-only control, review blocking and washes, and reduce detection exposure if needed (general IHC practice). Judge IL33 only where cell identity and localization remain clear (HPA tissue IHC).
A nominally negative tissue contains stained cells.	HPA's Not detected entries refer to specified cell types, such as duodenal glandular cells or kidney glomerular cells, not every cell in those tissues (HPA tissue IHC).	Identify the stained cell type before calling the result discordant. If the specified negative population stains strongly, investigate nonspecific detection with an appropriate control (HPA tissue IHC; general IHC practice).
Macrophages stain faintly while nearby epithelium is stronger.	Lung macrophages are listed Low, while selected respiratory epithelial populations are High (HPA tissue IHC); intensity depends on the sampled cell population.	Record macrophage and epithelial staining separately, including compartment and intensity (HPA tissue IHC). Avoid treating faint macrophage staining alone as proof of assay failure.
IF/ICC: what localization should be expected?	HPA reports supported nucleoplasmic localization with additional vesicles and lists ICC-IF images in BJ, EFO-21, and U2OS (HPA subcellular).	Use that localization as interpretation context and consult the separate IF/ICC guide for its workflow. The ICC-IF record does not supply an IHC-P protocol (HPA subcellular).

Source: UniProt O95760 Human Protein Atlas tissue Human Protein Atlas subcellular

Experiment evidence

IHC & IF images showing IL33 staining

IHC analysis of IL33 using anti-IL33 antibody (A00113). IL33 was detected in paraffin-embedded section of human tonsil . Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1ug/ml rabbit anti-IL33 Antibody (A00113) overnight at 4 Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37 The tissue section was developed using Strepavidin-Biotin-Complex (SABC)(Catalog # SA1022) with DAB as the chromogen.

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)

HPA · ICC-IF BJ [Human fibroblast]Human Protein Atlas immunofluorescence, BJ [Human fibroblast], antibody HPA022899; green = IL33, blue = nucleus, red = microtubules.

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Real IHC / IF data behind the staining-pattern expectations in this section.

Sample controls for IL33 IHC & IF

🧪Run bronchus first and require staining in respiratory epithelial cells (HPA: High in bronchus respiratory epithelial cells). Use adrenal gland glandular cells as the negative tissue (HPA: Not detected); on the bronchus slide, cells without specific staining should retain only the counterstain, but the HPA row does not establish which neighboring cell types are IL33-negative.

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) slide, a concentration-matched rabbit IgG isotype control, and IL33-knockout material as a biological specificity control (caption: rabbit primary antibody; standard IHC controls). For DAB detection, block endogenous peroxidase and check for endogenous biotin background because the selected caption uses a biotinylated secondary and streptavidin–biotin detection (selected A00113 caption; standard IHC practice).

⚠️Feasibility: No target-specific fixation window or fixation effect is reported, and the selected A00113 paraffin-section caption does not state the fixative (selected A00113 caption). Heat retrieval in citrate buffer at pH 6 for 20 minutes is documented for tonsil; its necessity in bronchus has not been established (selected A00113 caption). The supplied evidence does not establish that frozen sections or IF are easier; when interpreting bronchus DAB staining, assess peroxidase and biotin background with the controls above (selected A00113 caption; standard IHC practice).

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 section IHC using the catalog antibody’s tissue image, protein annotation, and cell specific staining evidence.

Which retrieval conditions should I start with when IL33 staining is weak?

Start with heat mediated citrate retrieval at pH 6 for 20 minutes in paraffin sections (caption A00113). Use the stained human tonsil section as a reference for that retrieval condition, while recording heating and cooling conditions so runs can be compared (caption A00113; standard IHC practice). If nuclear signal is weak, compare adjacent sections with a controlled change in retrieval time while keeping antibody concentration and detection conditions fixed (HPA tissue IHC; standard IHC practice). Check whether stronger retrieval improves nuclear staining in the expected cells without increasing diffuse tissue background or damaging morphology (HPA tissue IHC; standard IHC practice).

How should I assess whether fixation is masking IL33?

The catalog image documents staining in a paraffin embedded human tonsil section, but its caption does not state the fixative (caption A00113). Target specific fixation sensitivity is therefore unknown; do not infer it from the reported tissue staining pattern or protein annotations (caption A00113; HPA tissue IHC; UniProt O95760). Compare sections with documented fixation histories under identical citrate pH 6 retrieval and detection conditions, scoring nuclear signal alongside tissue preservation (caption A00113; standard IHC practice). If results differ, report the fixation histories and staining change as an experimental observation rather than assigning an IL33 specific masking mechanism (standard IHC practice).

Should IL33 stain nuclei, cytoplasm, or extracellular spaces in tissue?

Prioritize sharply localized nuclear staining in endothelial cells and the reported subsets of immune and epithelial cells when reviewing chromogenic sections (HPA tissue IHC). IL33 is also annotated in the cytoplasm, secretory vesicles, and extracellular space, so nonnuclear signal requires careful context rather than automatic rejection (UniProt O95760). Compare the distribution with adjacent nuclei and tissue architecture; the supported nucleoplasmic location and additional vesicle location provide useful compartment references (HPA subcellular; standard IHC practice). Score nuclear and nonnuclear staining separately, and avoid calling diffuse interstitial DAB positive cells without an identifiable stained cell or convincing extracellular pattern (HPA tissue IHC; UniProt O95760; standard IHC practice).

Could IL33 processing or isoforms explain different staining patterns?

IL33 has 4 annotated isoforms and a full length 1–270 chain plus processed chains beginning at residues 95, 99, or 109 (UniProt O95760). An antibody recognizing the N terminal region could detect a different set of forms from one recognizing a retained C terminal region, but the supplied caption does not map this antibody’s epitope (UniProt O95760; caption A00113). Review any available epitope documentation before interpreting a change in nuclear versus extracellular DAB staining as processing (UniProt O95760; standard IHC practice). Report the stained compartment and cell type without claiming that chromogenic intensity distinguishes individual isoforms or cleavage products (UniProt O95760; standard IHC practice).

How can IF help resolve ambiguous IL33 staining in the IHC section?

Use a matched IF section to test whether IL33 signal overlaps nuclei and a marker identifying the expected endothelial, immune, or epithelial cell population (HPA tissue IHC; standard IF practice). Choose a fluorophore channel after inspecting tissue autofluorescence, and compare single stain controls before interpreting apparent colocalization (standard IF practice). Because IL33 has supported nucleoplasmic localization and no annotated transmembrane segment, permeabilize fixed cells sufficiently for access to intracellular antigen, while avoiding an assumed extracellular epitope orientation (HPA subcellular; UniProt O95760; standard IF practice). Reconcile the IF pattern with cell resolved DAB staining, since IF evidence alone does not establish how the catalog antibody performs under the section’s chromogenic conditions (standard IHC/IF practice).

How do I distinguish diffuse IL33 signal from chromogenic background?

Begin with a section omitting the primary antibody to identify signal from the secondary and chromogenic detection system (standard IHC practice). The demonstrated workflow used 10% goat serum blocking, 1 µg/ml primary antibody, a biotinylated secondary, and DAB development (caption A00113). Check endogenous peroxidase blocking and, for a biotin based detection system, assess endogenous biotin where background persists; treat these as general workflow checks (caption A00113; standard IHC practice). Compare diffuse staining with crisp nuclear signal in annotated cell populations, and inspect edges and damaged regions before attributing brown deposits to IL33 (HPA tissue IHC; standard IHC practice).

What should I quantify when IL33 staining varies across cell populations? ⚠ ANSWER MARKED FOR VERIFICATION

Define the compartment and cell population before analysis: nuclear endothelial staining and staining in the reported immune or epithelial subsets may represent different readouts (HPA tissue IHC). Record % positive cells and an H score within each defined population, using the number of eligible cells as the denominator (standard IHC practice). If studying spatial abundance, report IL33 positive cell density per mm² of evaluable tissue and document excluded damaged or empty areas (standard IHC practice). Keep retrieval, DAB development, counterstain, imaging, and positivity thresholds consistent across sections, and report nuclear and nonnuclear scores separately (caption A00113; HPA tissue IHC; standard IHC practice).

What evidence makes a tissue IL33 positive call convincing?

A convincing positive call has reproducible cell resolved staining in an expected compartment, especially nuclei of the reported endothelial, immune, or epithelial subsets (HPA tissue IHC). Compare with a primary omission control and inspect section edges, necrotic areas, and endogenous enzyme signal before accepting brown deposits (standard IHC practice). Do not infer IL33 from staining in an unexpected cell solely because the surrounding tissue has IL33 RNA; secreted protein can complicate protein and RNA correspondence (HPA tissue IHC; UniProt O95760). Document compartment, cell identity, morphology, and control results, then classify uncertain diffuse extracellular staining separately from confident nuclear positivity (HPA tissue IHC; UniProt O95760; standard IHC practice).

Boster reagents

Best IL33 / Interleukin-33 IHC Antibodies

A00113 has a human tonsil paraffin-section IHC image (A00113 image caption). No IF image is supplied (catalog: A00113 IF image set).

Real IHC data

Anti-IL33 Antibody ®

Cat # A00113

View product →

A00113 is listed for human IHC, and its image shows IL33 staining in a paraffin section of human tonsil (catalog: A00113 applications/reactivity; A00113 image caption). A00113 has no listed IF/ICC application or IF image (catalog: A00113 applications/IF image set).

Which to pick: Choose A00113 for human paraffin-section chromogenic IHC: its caption documents citrate retrieval at pH 6 for 20 minutes and DAB detection; the fixative is unreported (A00113 image caption). There is no supported IF/ICC pick because A00113 has no listed IF/ICC application or IF image (catalog: A00113 applications/IF image set). There is no supported cross-species pick because A00113 lists Human only; its clone is unspecified (catalog: A00113 reactivity/clone).

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.