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IL23A / Interleukin-23 subunit alpha · IHC design guide
Design Immunohistochemistry for IL23A
Plan chromogenic IHC-P for IL23A using formaldehyde-fixed paraffin sections with catalog antibody A01097 (datasheet A01097). Use lymph node non-germinal center cells as a high-staining reference and assess cytoplasmic signal in immune-cell subsets (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
IL23A 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 | Cytoplasmic staining in immune-cell subsets (HPA tissue IHC) | |
| Staining pattern | Immune-cell subsets show cytoplasmic staining (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6 HIER, heat-mediated (datasheet A01097) | |
| Positive control | Lymph node+4 more · see all | |
| Negative control | Adipose tissue+4 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Formaldehyde-fixed paraffin sections (datasheet A01097, tissue IHC) (selected-SKU IHC image A01097) | |
| Caveat | Secretion may separate IL23A from its source cells (UniProt) | |
| Regulation | Expressed by activated dendritic and phagocytic cells (UniProt) | |
| Isoform / epitope | No isoforms; map epitopes to mature aa 20–189 (UniProt) |
Section 1
Recommended IL23A IHC & IF Protocols
The catalog antibody protocol specifies citrate pH 6 heat retrieval (datasheet A01097). A published IL23A IHC protocol provides incubation and detection details for colorectal cancer tissue (PMC8129521).
| Sample | Formaldehyde-fixed, paraffin-embedded human pancreas tissue (datasheet A01097) |
| Fixation | Image formalin-fixed; duration unreported (datasheet A01097); 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 (datasheet A01097); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% serum (datasheet A01097) |
| Primary antibody | Rabbit anti-IL23A, 5 μg/mL (datasheet A01097) |
| Primary incubation | Overnight at 4 °C (standard) |
| Detection | HRP-polymer secondary, DAB chromogen 5–10 min (standard) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | IL23A-positive staining in non-germinal center cells of lymph node (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression mainly in subsets of immune cells. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6 heat retrieval (datasheet A01097); the published protocol does not specify a retrieval method (PMC8129521).
Section 2
What Is the Expected IL23A Staining Pattern?
IL23A should appear mainly in the cytoplasm of subsets of immune cells in tissue sections (HPA tissue IHC: Approved; cytoplasmic immune-cell profile). Lymph-node non-germinal-center cells provide a reported high-staining example (HPA tissue IHC: High). IL23A has no transmembrane segment and is secreted upon association with IL12B (UniProt Q9NPF7: topology and subunit). HPA cautions that secreted-protein RNA and protein locations may differ and reports disregarded presumed off-target binding (HPA tissue IHC: reliability description).
| Discrete cytoplasmic staining in lymph-node non-germinal-center cells, with other cells less conspicuous (HPA tissue IHC: High in non-germinal-center cells). | This fits the reported tissue pattern, especially if the same cell population remains distinct from background (HPA tissue IHC: cytoplasmic immune-cell profile; standard IHC practice). |
| Predominantly nuclear or sharply membrane-bound staining, without a convincing cytoplasmic component (HPA tissue IHC: cytoplasmic profile). | Treat this as discordant and investigate staining artefact or antibody specificity; IL23A has no transmembrane segment (UniProt Q9NPF7: topology; standard IHC practice). |
| Strong staining in bronchial respiratory epithelium while the expected immune-cell pattern is absent (HPA tissue IHC: respiratory epithelial cells not detected). | Consider cross-reactivity or endogenous detection activity, and compare with a negative reagent control (HPA tissue IHC: presumed off-target binding disregarded; standard IHC practice). |
| A uniform haze covers cells and surrounding tissue, obscuring their boundaries (standard IHC practice). | Diffuse background cannot establish IL23A-positive cells; check blocking, antibody concentration, washing and detection controls before scoring (standard IHC practice). |
| No convincing signal in lymph-node non-germinal-center cells (HPA tissue IHC: High in these cells). | First assess section quality and the detection workflow; absence in one preparation does not overturn the reported tissue pattern (HPA tissue IHC: High; standard IHC practice). |
💡Expected IL23A appearanceA credible positive is distinct cytoplasmic staining in a subset of immune cells, potentially high in lymph-node non-germinal-center cells; broad nuclear, membrane-only or uniform background staining warrants review (HPA tissue IHC: profile and High; UniProt Q9NPF7: topology; standard IHC practice).
| Secretion and assembly (UniProt Q9NPF7: subcellular location and subunit). | IL23A is secreted upon association with IL12B, so cytoplasmic tissue staining is compatible with cellular production; an extracellular deposit alone cannot identify the producing cell (UniProt Q9NPF7: secretion; standard IHC practice). |
| Signal-peptide processing (UniProt Q9NPF7: residues 1–19 and mature chain 20–189). | The precursor has a signal peptide and the annotated chain begins at residue 20; the supplied evidence gives no antibody epitope, so it cannot predict recognition of either form (UniProt Q9NPF7: processing; supplied antibody record). |
| Tissue and cell context (HPA tissue IHC: positive and negative observations). | HPA reports High staining in lymph-node non-germinal-center cells and Medium staining in appendix lymphoid tissue, marrow hematopoietic cells, tonsil germinal-center cells and testis spermatids. Adipocytes are among the listed cells with no detection (HPA tissue IHC: stated levels). |
| Scope of validation (HPA tissue IHC: reliability Approved; HPA001554 IHC Approved). | The reported pattern is a useful reference, but HPA also notes disregarded presumed off-target binding. Its supplied antibody status is Approved, with no Enhanced status given (HPA tissue IHC: reliability description; HPA antibodies: HPA001554). |
| Situation | Likely cause | Next action |
|---|---|---|
| The expected lymph-node cell population is blank (HPA tissue IHC: High in non-germinal-center cells). | A failed staining run or unsuitable section may hide the expected pattern (standard IHC practice). | Check section integrity and the run's positive and detection controls, then review the catalog antibody's IHC-P instructions before repeating (standard IHC practice). |
| Brown signal is diffuse across the section (standard IHC practice). | Background from blocking, antibody concentration, washing or detection can obscure cell-specific staining (standard IHC practice). | Compare a negative reagent control; optimize blocking, dilution and washes while preserving a known-positive reference section (standard IHC practice; HPA tissue IHC: lymph-node High). |
| Staining appears chiefly nuclear or membrane-bound (HPA tissue IHC: cytoplasmic profile). | That distribution conflicts with the reported tissue pattern and the absence of a transmembrane segment (HPA tissue IHC: profile; UniProt Q9NPF7: topology). | Recheck the cell boundaries and counterstain, then assess antibody and detection controls before calling the pattern specific (standard IHC practice). |
| Bronchial respiratory epithelial cells stain strongly (HPA tissue IHC: not detected). | The result may reflect cross-reactivity or endogenous detection activity; HPA reports disregarded presumed off-target binding (HPA tissue IHC: reliability description; standard IHC practice). | Compare a negative reagent control and an expected positive tissue, and avoid scoring epithelial signal alone as IL23A (HPA tissue IHC: lymph-node High; standard IHC practice). |
| A positive cell appears outside the main immune-cell pattern (HPA tissue IHC: cytoplasmic immune-cell profile). | Location alone is inconclusive: HPA also reports Medium staining in round or early spermatids, and UniProt lists keratinocytes among secretory cells (HPA tissue IHC: testis Medium; UniProt Q9NPF7: tissue specificity). | Identify the cell and compartment first, then compare that exact population with the supplied tissue observations and controls (HPA tissue IHC: cell-level observations; standard IHC practice). |
| Does an ICC-IF image confirm an IHC-P result? (HPA subcellular: ICC-IF cytosol Approved). | ICC-IF reports cytosolic localisation, while tissue IHC has its own Approved staining profile and noted off-target concern (HPA subcellular: cytosol Approved; HPA tissue IHC: reliability description). | Use ICC-IF as a compartment cross-check only; judge the paraffin-section result against tissue cell patterns and IHC controls (HPA subcellular: cytosol; HPA tissue IHC: profile; standard IHC practice). |
Experiment evidence
IHC & IF images showing IL23A staining
A01097 · IHCImmunohistochemistry Validation of IL-23 in Human Pancreas Tissue Immunohistochemical analysis of paraffin-embedded human pancreas tissue us
A01097 · IHCImmunohistochemistry Validation of IL-23 in Human Abdominal Aortic Aneurysm (AAA) Tissues (Yan et al., 2019)
A01097 · IFImmunofluorescence Validation of IL-23 in Raji Cells Immunofluorescent analysis of 4% paraformaldehyde-fixed Raji Cells labeling IL-23 with
A01097-2 · IHCImmunohistochemistry Validation of IL-23 in Mouse Pancreas Tissue Immunohistochemical analysis of paraffin-embedded mouse pancreas tissue us
A01097-2 · IHCInduced Expression Validation of IL-23 in Mouse Keratinocytes of Epidermis (Yoon et al., 2016) The expression of IL-23 protein induced by ta
A01097-2 · IFImmunofluorescence Validation of IL-23 in Mouse Pancreas Tissue Immunofluorescent analysis of 4% paraformaldehyde-fixed mouse pancreas tissu
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000110944-IL23A/tissue/cerebral+cortex#imid_598368
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000110944-IL23A/tissue/colon#imid_598310
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000110944-IL23A/tissue/liver#imid_598303
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000110944-IL23A/tissue/kidney#imid_598296
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA001554; green = IL23A, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA001554; green = IL23A, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for IL23A IHC & IF
🧪Run lymph node first; non-germinal center cells should stain (HPA: High in lymph-node non-germinal center cells). Use adipose tissue as a negative and expect adipocytes to lack staining (HPA: Not detected in adipocytes); on the lymph-node slide, use adjacent unstained cells as internal negative comparators without assigning them a specific cell identity.
Positive control tissue: Lymph node (Non-germinal center cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show IL23A in A-431, U-251MG, U2OS, with annotated localisation: Cytosol (approved) (HPA subcellular).
Technical controls: Include a no-primary (secondary-only) control, an isotype control matched to the primary antibody’s host species and immunoglobulin class, and IL23A-knockout material as a biological negative. For chromogenic detection in lymph node, quench endogenous peroxidase and assess residual background on the no-primary slide.
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported; the A01097 caption mentions formaldehyde but gives no fixation duration, while its separate fixative field says “not stated in the caption” (A01097 tissue-IHC caption). The paraffin-section example uses heat retrieval in citrate buffer at pH 6, but it does not establish that retrieval is required (A01097 tissue-IHC caption). The supplied evidence does not establish whether frozen sections or IF are easier; because IL23A is secreted upon association with IL12B, interpret diffuse lymph-node signal cautiously alongside cellular staining and controls (UniProt Q9NPF7 subcellular; HPA: High in lymph-node non-germinal center cells).
HPA tissue IHC evidence for IL23A
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Secreted protein, tissue location of RNA and protein is expected to differ. Presumed off target binding observed and disregarded.). 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 |
|---|---|---|---|---|
| Lymph node | Non-germinal center cells | High | Protein (IHC) | HPA → |
| Appendix | Lymphoid tissue | Medium | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | Medium | Protein (IHC) | HPA → |
| Testis | Round or early spermatids | Medium | Protein (IHC) | HPA → |
| Tonsil | Germinal center cells | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Adrenal gland | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Breast | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | Not detected | Protein (IHC) | HPA → |
| Caudate | Glial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced IL23A IHC Tips
Troubleshoot IL23A staining in paraffin sections by checking retrieval, cellular context and controls before interpreting chromogenic signal.
Which retrieval conditions should I start with for IL23A paraffin IHC?
Use heat-mediated citrate buffer at pH 6 for antigen retrieval in paraffin sections (datasheet A01097). The selected tissue IHC image used this retrieval with the catalog antibody at 5 µg/ml, followed by overnight primary incubation at 4°C (datasheet A01097). If staining is weak, compare a modest change in heating time with the original condition on adjacent sections, while keeping antibody concentration and detection constant (standard IHC practice). Inspect tissue structure and background after each change, because excessive heating can impair morphology and make a gain in apparent staining difficult to interpret (standard IHC practice).
How should I investigate weak staining after fixation?
Target-specific fixation sensitivity for IL23A is unknown from the supplied evidence; do not infer it from tissue staining patterns or protein topology (supplied evidence). Start by comparing sections with documented, consistent fixation and processing histories, then apply the same citrate pH 6 retrieval and detection steps to each (datasheet A01097; standard IHC practice). Use adjacent sections and a positive control in the same staining run to distinguish a processing problem from a run-wide failure (standard IHC practice). Record fixation duration and section condition before changing retrieval or antibody concentration, since changing several variables together prevents a clear diagnosis (standard IHC practice).
Where should convincing IL23A staining appear in tissue?
Expect mainly cytoplasmic staining in subsets of immune cells in tissue sections (HPA: tissue IHC profile). IL23A has a signal peptide at residues 1–19, lacks a transmembrane segment, and is secreted upon association with IL12B, so an exclusively crisp membrane pattern needs independent validation (UniProt Q9NPF7). Compare candidate cells with local tissue morphology and, where feasible, a serial section stained for the suspected cell population (standard IHC practice). Diffuse extracellular signal may be biologically plausible for a secreted protein, but it cannot by itself identify the producing cell; score cellular and extracellular staining separately (UniProt Q9NPF7; standard IHC practice).
Could processing or IL-23 assembly alter what this antibody detects?
The supplied record lists one mature IL23A chain, residues 20–189, after removal of the 1–19 signal peptide, and lists no alternative isoforms or annotated glycosylation sites (UniProt Q9NPF7). IL23A forms a disulfide-linked heterodimer with IL12B, so epitope accessibility could differ between free and assembled protein; the supplied evidence does not map this antibody’s epitope (UniProt Q9NPF7; supplied evidence). Avoid interpreting staining intensity as a direct measure of intact IL-23 without separate evidence of IL12B association (UniProt Q9NPF7; standard IHC interpretation). If results conflict with expected cell distribution, seek independent antibody or orthogonal evidence before assigning the signal to a specific molecular form (standard IHC practice).
How can IF help check an ambiguous chromogenic IL23A pattern?
For a separate IF experiment, pair IL23A with a marker for macrophages or dendritic cells, which are described as IL23A-producing populations, and assess overlap cell by cell (UniProt Q9NPF7; standard IF practice). Select a red or far-red fluorophore after checking unstained tissue for autofluorescence in each channel (standard IF practice). For intracellular IL23A, use controlled permeabilisation to expose the cytosolic epitope; compare a nonpermeabilised section if extracellular signal is also of interest (HPA: approved cytosolic location; UniProt Q9NPF7; standard IF practice). Include single-stain and secondary-only controls to check channel bleed-through and nonspecific fluorescence before comparing IF with chromogenic IHC (standard IF practice).
What should I adjust when IL23A staining is widespread or granular?
First compare the full stain with a no-primary control and check whether signal follows tissue edges, damaged areas or multiple unrelated cell types (standard IHC practice). Block endogenous peroxidase for an HRP chromogenic workflow and confirm that the block itself preserves readable tissue morphology (standard IHC practice). The selected tissue IHC image used 10% serum for 1 hour at room temperature and a primary concentration of 5 µg/ml; use these as documented starting conditions, then titrate if background persists (datasheet A01097; standard IHC practice). Treat broad staining cautiously because the HPA tissue profile explicitly notes presumed off-target binding (HPA: reliability description).
How should I quantify IL23A IHC across samples? ⚠ ANSWER MARKED FOR VERIFICATION
Define the tissue region and eligible cell population before scoring, then report either an H-score for cellular intensity and prevalence or the percentage of positive cells (standard IHC practice). For sparse immune cells, positive-cell density per mm² can complement percentage positive, provided each section uses the same area-selection rule (standard IHC practice). Normalise counts to the number of eligible cells or the measured viable tissue area, and report cellular staining separately from diffuse extracellular signal (standard IHC practice; UniProt Q9NPF7: secreted). Use matched exposure, chromogen development and positivity thresholds across the comparison, and exclude damaged or necrotic regions by a predefined rule (standard IHC practice).
How can I distinguish a true IL23A-positive cell from artefact?
A credible cellular signal should fit a cytoplasmic pattern and the morphology of a plausible producing cell, such as an activated dendritic or phagocytic cell (HPA: tissue IHC profile; UniProt Q9NPF7: tissue specificity). Be skeptical of uniform nuclear or sharp membrane staining, especially when it also appears in the no-primary control (HPA: approved cytosolic location; UniProt Q9NPF7: no transmembrane segment; standard IHC practice). Check section edges and necrotic areas for nonspecific deposition, and compare suspected staining with the endogenous-peroxidase control in the HRP workflow (standard IHC practice). Finally, require independent support for surprising cell assignments because the HPA assessment reports presumed off-target binding (HPA: reliability description).
Boster reagents
Best IL23A / Interleukin-23 subunit alpha IHC Antibodies
Validated anti-IL23A antibodies have IHC images from human and mouse pancreas sections and IF images from human and mouse samples (catalog: A01097 and A01097-2 image captions).
Real IHC data
Anti-IL-23 Antibody
Cat # A01097
Real IHC data
Anti-IL-23 Antibody
Cat # A01097-2
A01097 has IHC data from paraffin-embedded human pancreas and IF data from human pancreas and Raji cells (catalog: A01097 image captions). A01097-2 has IHC and IF data from mouse pancreas, with Human and Mouse listed as reactive species (catalog: A01097-2 image captions and reactivity).
Which to pick: For human tissue IHC, choose A01097 at 5 μg/mL; its paraffin-section image reports formaldehyde fixation (catalog: A01097 IHC caption). For IF or ICC in human samples, choose A01097, which lists both applications and shows human IF and ICC images (catalog: A01097 applications and image captions). For work spanning human and mouse, consider A01097-2: both species are listed, but its IHC and IF images show mouse samples, and its paraffin-section IHC image reports formaldehyde fixation and 2 μg/mL; clone type is unspecified for both SKUs.
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q9NPF7 (IL23A_HUMAN, Interleukin-23 subunit alpha).
- Human Protein Atlas. IL23A tissue IHC expression (reliability: Approved).
- Human Protein Atlas. IL23A subcellular location (ICC-IF): Localized to the cytosol..
- Human Protein Atlas. IL23A antibody validation summary (1 antibodies).
- Identification and Validation of a Six Immune-Related Genes Signature for Predicting Prognosis in Patients With Stage II Colorectal Cancer. Frontiers in genetics 2021 — PMC8129521.
- Blocking IL-23 Signaling Mitigates Cigarette Smoke-Induced Murine Emphysema. Environmental toxicology 2024 — PMC11567802.
- Cytokine RNA In Situ Hybridization Permits Individualized Molecular Phenotyping in Biopsies of Psoriasis and Atopic Dermatitis. JID innovations : skin science from molecules to population health 2021 — PMC8659380.
- Role of macrophage CCAAT/enhancer binding protein delta in the pathogenesis of rheumatoid arthritis in collagen-induced arthritic mice. PloS one 2012 — PMC3454428.
- PubMed PMID:11114383 — UniProt-cited evidence.
- PubMed PMID:12975309 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for IL23A (Interleukin-23 subunit alpha). Expected localisation Cytoplasmic staining in immune-cell subsets (HPA tissue IHC). Antigen retrieval: Citrate pH 6 HIER, heat-mediated (datasheet A01097). Validated IHC antibody: A01097 at 5 μg/mL (the product's own IHC validation image is shown). Controls (HPA): positive Lymph node (Non-germinal center cells); negative Adipose tissue. IHC-P workflow on paraffin sections. Protocol steps: 1. Sample: Formaldehyde-fixed, paraffin-embedded human pancreas tissue (datasheet A01097); 2. Fixation: Image formalin-fixed; duration unreported (datasheet A01097); 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 (datasheet A01097); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% serum (datasheet A01097); 8. Primary antibody: Rabbit anti-IL23A, 5 μg/mL (datasheet A01097); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Formaldehyde-fixed paraffin sections (datasheet A01097, tissue IHC) (selected-SKU IHC image A01097); Caveat: Secretion may separate IL23A from its source cells (UniProt); Isoform / epitope: No isoforms; map epitopes to mature aa 20–189 (UniProt); Regulation: Expressed by activated dendritic and phagocytic cells (UniProt). Evidence: UniProt Q9NPF7, HPA tissue IHC · Approved, HPA ICC-IF · cytosol., Datasheet A01097, PMC8129521. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/IL23A/immunohistochemistry-ihc-guide.