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FOXA3 / Hepatocyte nuclear factor 3-gamma · IHC design guide
Design Immunohistochemistry for FOXA3
Plan FOXA3 paraffin IHC around nuclear staining and a catalog antibody concentration of 2–5 μg/mL (HPA tissue IHC; datasheet PB9805). Include controls to assess specificity because HPA rates the tissue staining evidence uncertain (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
FOXA3 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; tissue evidence is uncertain (HPA tissue IHC) | |
| Staining pattern | General nuclear staining across tissue cells (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet PB9805) | |
| Positive control | Adipose tissue+4 more · see all | |
| Negative control | Lymph node |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent across sections (standard IHC practice; not target-specific) | |
| Caveat | Low staining–RNA consistency warrants validation (HPA tissue IHC) | |
| Regulation | Expression regulation is unreported (UniProt) | |
| Isoform / epitope | No annotated isoforms; one 1–350 chain (UniProt) |
Section 1
Recommended FOXA3 IHC & IF Protocols
The catalog antibody protocol uses EDTA pH 8.0 retrieval (datasheet: PB9805). Published FOXA3 IHC protocols below provide three tissue staining examples (PMC7957975; PMC7084256; PMC2681412).
| Sample | Paraffin-embedded human colon cancer tissue; fixative not specified (datasheet PB9805) |
| Fixation | Image fixative and duration unreported (datasheet PB9805); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: EDTA pH 8.0 (datasheet PB9805); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet PB9805) |
| Primary antibody | Rabbit anti-FOXA3, 2-5 μg/ml (datasheet PB9805) |
| Primary incubation | Overnight at 4 °C (datasheet PB9805) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet PB9805) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | FOXA3-positive staining in adipocytes of adipose tissue (HPA tissue IHC: High). HPA tissue profile: General nuclear expression. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA pH 8.0 for the catalog antibody (datasheet: PB9805). The published protocols describe citrate retrieval for other antibodies (PMC7957975; PMC7084256; PMC2681412).
Section 2
What Is the Expected FOXA3 Staining Pattern?
FOXA3 is a nuclear transcription factor with no transmembrane segment (UniProt P55318: nucleus; topology). Expect nuclear staining in cells reported positive by HPA, such as bronchial respiratory epithelial cells and lung alveolar cells (HPA tissue IHC: High). Treat these as candidate controls: HPA calls the tissue IHC profile Uncertain because antibody staining has low consistency with RNA expression and awaits external verification (HPA tissue IHC: reliability).
| Discrete nuclear chromogen in bronchial respiratory epithelial cells or lung alveolar cells. | This matches the reported compartment and cell patterns (UniProt P55318: nucleus; HPA tissue IHC: High in both). Compare cell by cell with the counterstain and a control section. The apparent strength alone does not confirm specificity because the overall tissue IHC profile is Uncertain (HPA tissue IHC: reliability). |
| Predominantly cytoplasmic or membrane-like staining, with little nuclear signal. | The compartment disagrees with FOXA3 localisation (UniProt P55318: nucleus; HPA ICC-IF: supported nucleoplasm). Treat it as a possible artefact and review detection and control staining before scoring it as FOXA3. UniProt reports no transmembrane segment, so a membrane outline is especially difficult to reconcile with the target (UniProt P55318: topology). |
| Strong staining in lymph-node germinal-center cells. | These cells are reported as not detected by HPA tissue IHC (HPA tissue IHC: lymph node). Consider cross-reactivity or endogenous detection activity; check the no-primary control and whether the signal is truly nuclear. Because HPA rates its tissue IHC profile Uncertain, this comparison is a warning sign, not proof that every positive cell is false (HPA tissue IHC: reliability). |
| Widespread haze or precipitate obscures nuclei and tissue boundaries. | A diffuse deposit cannot establish nuclear FOXA3 localisation (UniProt P55318: nucleus). In general IHC practice, review blocking, washes, detection background and chromogen development against a no-primary control. Score only cells whose nuclear signal can be distinguished from the surrounding background. |
| No nuclear signal in a bronchus or lung candidate control. | HPA reports High staining in bronchial respiratory epithelial cells and lung alveolar cells, making them useful comparison candidates (HPA tissue IHC: bronchus; lung). First verify that the expected cells are present, then review the IHC-P run and antibody performance. A blank section alone cannot resolve whether the assay failed or the uncertain reference pattern failed to reproduce (HPA tissue IHC: reliability). |
💡Expected FOXA3 appearanceA plausible positive is distinct nuclear chromogen in an HPA-reported High cell population, such as bronchial respiratory epithelium; broad cytoplasmic, membrane-like or control-matched deposit is suspect (UniProt P55318: nucleus; HPA tissue IHC: bronchus, High; reliability Uncertain).
| Localisation and topology | FOXA3 is annotated in the nucleus and has no transmembrane segment (UniProt P55318: subcellular location; topology). Interpret IHC-P signal by its alignment with nuclei; membrane outlines or isolated cytoplasmic colour require scrutiny. Supported nucleoplasmic localisation in HPA ICC-IF offers a separate compartment check, without establishing an IHC-P staining intensity (HPA subcellular: nucleoplasm, supported). |
| Tissue evidence and control selection | HPA reports High IHC staining in several cell populations but labels the overall tissue profile Uncertain because staining and RNA expression show low consistency (HPA tissue IHC: positive entries; reliability). UniProt reports expression in liver, pancreas, erythroleukemia and hepatoma cell lines, with no expression in other examined samples (UniProt P55318: tissue specificity). Use the reported IHC-positive cells as candidate controls and avoid treating broad staining as independently validated. |
| Antibody evidence | The supplied antibody record marks CAB015154 IHC as Uncertain and HPA054034 ICC as Supported (HPA antibodies: CAB015154; HPA054034). Those ratings apply to different assays. An ICC rating cannot substitute for IHC-P validation; assess any IHC result with its own positive and no-primary controls (general IHC practice). |
| IF/ICC Q: What pattern should be expected? | A: HPA reports mainly nucleoplasmic localisation, supported in ICC-IF, with images from CACO-2, Hep-G2, U2OS and KOLF2.1J (HPA subcellular: summary; image cell lines). Use the separate IF/ICC guide for assay conditions; this observation does not provide an IF/ICC protocol or establish staining in every listed cell (HPA subcellular: ICC-IF images). |
| Situation | Likely cause | Next action |
|---|---|---|
| Positive candidate tissue is blank. | The expected cells may be absent from the section, or the IHC-P run may have failed; HPA's positive tissue profile is also Uncertain (HPA tissue IHC: reliability). | Confirm tissue identity and expected cell morphology, then compare an IHC-P positive control processed in the same run. Review the catalog antibody's IHC-P instructions before changing retrieval or dilution (general IHC practice). |
| Only cytoplasm or cell borders stain. | That distribution conflicts with the nuclear target and supported nucleoplasmic ICC-IF location (UniProt P55318: nucleus; HPA subcellular: nucleoplasm). | Check the counterstain to locate nuclei, inspect the no-primary control and review whether detection deposits or tissue edges explain the colour. Do not score compartment-mismatched staining as a confirmed FOXA3 positive (general IHC practice). |
| Lymph-node germinal-center cells stain strongly. | HPA reports these cells as not detected, so the result conflicts with that reference pattern; the HPA tissue profile itself remains Uncertain (HPA tissue IHC: lymph node; reliability). | Compare with a no-primary control and examine whether staining is nuclear. Repeat with an independently validated IHC antibody or orthogonal evidence if the unexpected result is central to the study (general IHC practice). |
| All sections show diffuse brown background. | General IHC sources include nonspecific antibody or detection binding, endogenous detection activity and excess chromogen development (general IHC practice). | Run a no-primary control, review blocking and washing, and inspect chromogen development. Reassess only after individual nuclei are distinguishable from the background (general IHC practice). |
| Two antibody results disagree. | The supplied HPA antibody ratings cover different assays: CAB015154 is IHC Uncertain, while HPA054034 is ICC Supported (HPA antibodies: CAB015154; HPA054034). | Compare results only within the same application and check each antibody's own validation and controls. Use independent IHC evidence before attributing a discrepant chromogenic pattern to FOXA3 (general IHC practice). |
| Broad nuclear staining seems inconsistent with expression data. | HPA describes general nuclear IHC expression but low agreement with RNA, whereas UniProt describes a more restricted expression set (HPA tissue IHC: profile; reliability; UniProt P55318: tissue specificity). | Record the cell types and compartments separately from staining intensity. Report the discrepancy and seek independent evidence before calling the broad pattern target-specific (general IHC practice). |
Experiment evidence
IHC & IF images showing FOXA3 staining
PB9805 · IHCIHC analysis of FOXA3 using anti-FOXA3 antibody (PB9805). FOXA3 was detected in a paraffin-embedded section of human colon cancer tissue. He
PB9805 · IHCIHC analysis of FOXA3 using anti-FOXA3 antibody (PB9805). FOXA3 was detected in a paraffin-embedded section of human pancreatic cancer tissu
PB9805 · IFIF analysis of SDHA using anti-SDHA antibody (PB9433). SDHA was detected in an immunocytochemical section of U2OS cells. Enzyme antigen retr
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000170608-FOXA3/tissue/cerebral+cortex#imid_3967208
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000170608-FOXA3/tissue/colon#imid_3967126
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000170608-FOXA3/tissue/liver#imid_3967135
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000170608-FOXA3/tissue/kidney#imid_3967144
HPA · ICC-IF CACO-2Human Protein Atlas immunofluorescence, CACO-2, antibody HPA054034; green = FOXA3, blue = nucleus, red = microtubules.
HPA · ICC-IF CACO-2Human Protein Atlas immunofluorescence, CACO-2, antibody HPA054034; green = FOXA3, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for FOXA3 IHC & IF
🧪Run bronchus first: respiratory epithelial cells should show nuclear staining (HPA: High in respiratory epithelial cells; UniProt P55318: Nucleus). Use lymph node germinal center cells as the negative tissue (HPA: Not detected in germinal center cells); on the bronchus slide, use nearby cells without nuclear staining as an internal background reference, without assuming every non-epithelial cell is FOXA3-negative.
Positive control tissue: Adipose tissue (Adipocytes, HPA High)
Negative control tissue: Lymph node (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show FOXA3 in CACO-2, Hep-G2, U2OS, KOLF2.1J, with annotated localisation: Nucleoplasm (supported) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control, nonimmune rabbit IgG matched to the primary's clonality if known, and a FOXA3 knockout specimen as a biological negative (selected PB9805 caption: rabbit primary antibody). For bronchus, quench endogenous peroxidase before chromogenic detection and check mucus-associated background (standard IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected PB9805 paraffin-section caption does not state a fixative (selected PB9805 caption). That caption uses heat-mediated retrieval in EDTA at pH 8.0, but does not establish that retrieval is required (selected PB9805 caption). Frozen sections and IF cannot be judged easier from the supplied evidence; for IF, assess autofluorescence separately, while bronchial mucus can complicate background assessment (standard IF/IHC practice).
HPA tissue IHC evidence for FOXA3
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Uncertain — Low consistency between antibody staining and RNA expression data. Pending external verification.). 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 |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | High | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | High | Protein (IHC) | HPA → |
| Caudate | Glial cells | High | Protein (IHC) | HPA → |
| Cerebral cortex | Glial cells | High | Protein (IHC) | HPA → |
| Heart muscle | Cardiomyocytes | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Lymph node | Germinal center cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced FOXA3 IHC Tips
Troubleshoot FOXA3 staining in paraffin sections by checking nuclear localisation, retrieval, controls and the limits of the available tissue evidence.
What retrieval should I try first when FOXA3 nuclear staining is weak?
Start with heat mediated antigen retrieval in EDTA at pH 8.0 for paraffin sections (datasheet PB9805). The selected tissue image used that retrieval before overnight primary incubation at 4°C, so keep those conditions aligned while assessing the result (datasheet PB9805). If nuclear staining remains weak, compare a shorter or longer heating period on matched sections, recording tissue preservation and background; the caption does not specify a retrieval duration (datasheet PB9805). Score nuclei in the same cell population across conditions, and include a no primary control so increased DAB deposition is not mistaken for recovered FOXA3 signal (UniProt P55318: nucleus).
How should I troubleshoot variable FOXA3 staining after fixation?
FOXA3 specific sensitivity to fixation is unknown: the selected paraffin tissue caption does not state its fixative (datasheet PB9805). Record the fixative, time before fixation and fixation duration for every specimen, then compare matched sections processed with the same retrieval and detection settings. If nuclear staining varies, examine morphology and the distribution of staining across section depth before changing antibody concentration; the documented primary concentration is 2 μg/ml with overnight incubation at 4°C (datasheet PB9805). Treat any improvement after changing fixation as a result of your own controlled comparison, and use nuclear localisation as the expected readout (UniProt P55318: nucleus).
How can I assess cytoplasmic or diffuse FOXA3 staining?
FOXA3 is annotated in the nucleus, and the supported subcellular location is the nucleoplasm (UniProt P55318: nucleus; HPA subcellular: nucleoplasm). Compare DAB signal with a nuclear counterstain at high magnification, recording the fraction of intact nuclei stained and whether cytoplasm or extracellular material also colours. For diffuse staining, repeat the assay with a no primary control and inspect tissue edges, folds and damaged areas before adjusting primary concentration or detection time. Keep the documented 2 μg/ml primary condition as a reference while troubleshooting (datasheet PB9805); a predominantly cytoplasmic pattern needs independent validation before attribution to FOXA3.
Could isoforms or epitope accessibility explain inconsistent FOXA3 staining?
The supplied record lists 0 isoforms, a single 1–350 chain, and no annotated glycosylation or modified residues (UniProt P55318 record). Those annotations do not identify the antibody epitope or establish how fixation and retrieval affect its accessibility, so compare matched sections under the documented EDTA pH 8.0 retrieval first (datasheet PB9805). If results still disagree, check the antibody documentation for its stated immunogen or epitope and seek an independently validated reagent recognising another region. Interpret persistent disagreement alongside morphology and nuclear localisation, without assigning it to an unlisted FOXA3 isoform (UniProt P55318: nucleus; UniProt P55318 record: 0 isoforms).
How should I check FOXA3 localisation in a multiplex IF follow-up?
For a separate IF experiment, pair FOXA3 with a validated marker for the expected cell population and a nuclear counterstain; FOXA3 should be assessed in nuclei (UniProt P55318: nucleus). Choose spectrally separated fluorophores and place the weaker signal in a channel with low measured tissue autofluorescence, checking unstained tissue and single stain controls. Because FOXA3 has no transmembrane segment and is mainly nucleoplasmic, include a permeabilisation step that allows antibody access to the nuclear compartment, then titrate it against cell preservation (UniProt P55318 topology; HPA subcellular: nucleoplasm). Keep IF results distinct from the paraffin section DAB image, which documents an IHC workflow for PB9805 (datasheet PB9805).
What should I check when DAB background obscures FOXA3 nuclei?
Run a no primary control alongside the test section to assess secondary reagent, endogenous peroxidase and chromogen contributions. The documented IHC image used goat serum blocking at 10%, a 2 μg/ml rabbit primary overnight at 4°C, and a peroxidase conjugated secondary for 30 minutes at 37°C before DAB development (datasheet PB9805). Peroxidase blocking, adequate washing and shorter chromogen development are general IHC adjustments; compare them on adjacent sections so weak nuclear signal is not lost. Record whether colour remains in nuclei or instead follows folds, damaged tissue or diffuse extracellular areas; the expected FOXA3 compartment is nuclear (UniProt P55318: nucleus).
How should I quantify FOXA3 staining across tissue sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and analysis region before scoring, then report the percentage of positive nuclei and, when intensity is reproducible, a nuclear H-score. Normalise counts to the number of evaluable nuclei in that population, or report positive nuclei per mm² with the analysed area and exclusion rules stated. Keep retrieval, 2 μg/ml primary concentration, development time and image settings consistent across specimens; the concentration comes from the selected IHC image (datasheet PB9805). Score background and no primary controls separately, and interpret differences cautiously because HPA rates its FOXA3 tissue staining reliability as uncertain owing to low agreement with RNA expression (HPA tissue IHC: uncertain).
When is a FOXA3 positive IHC result convincing?
A convincing result shows reproducible staining in intact nuclei, consistent with FOXA3's nuclear annotation and supported nucleoplasmic location (UniProt P55318: nucleus; HPA subcellular: nucleoplasm). Assess the stained cell population against morphology and an appropriate independent control; UniProt reports expression in liver and pancreas among the examined tissues (UniProt P55318: tissue specificity). Question colour confined to section edges, necrotic areas, cytoplasm or no primary controls, and assess endogenous peroxidase when interpreting DAB signal. The PB9805 caption documents staining in a paraffin section of human colon cancer, while HPA calls its broader tissue IHC reliability uncertain, so that image alone cannot establish specificity in every tissue (datasheet PB9805; HPA tissue IHC: uncertain).
Boster reagents
Best FOXA3 / Hepatocyte nuclear factor 3-gamma IHC Antibodies
PB9805 has FOXA3 paraffin-section IHC images from human colon and pancreatic cancers and mouse and rat liver (PB9805 IHC captions). IF/ICC is listed, but the supplied IF caption depicts SDHA (catalog IF caption).
Real IHC data
Anti-FOXA3 Antibody ®
Cat # PB9805
PB9805 is listed for IHC in human, mouse and rat, with paraffin-section images of human colon cancer, human pancreatic cancer, mouse liver and rat liver (catalog applications/reactivity; PB9805 IHC captions). PB9805 is also listed for IF/ICC, but its supplied IF caption identifies SDHA and PB9433, so it does not establish FOXA3 IF performance (catalog applications; catalog IF caption).
Which to pick: Choose PB9805 for paraffin-section tissue IHC, supported by its own FOXA3 images; the fixative is unreported (PB9805 IHC captions). For IF/ICC, PB9805 is the listed option, although its supplied IF image documents SDHA/PB9433 (catalog applications; catalog IF caption). For cross-species work, PB9805 lists human, mouse and rat reactivity; it is rabbit-hosted, and clonality is unreported (catalog reactivity/host/clone).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P55318 (FOXA3_HUMAN, Hepatocyte nuclear factor 3-gamma).
- Human Protein Atlas. FOXA3 tissue IHC expression (reliability: Uncertain).
- Human Protein Atlas. FOXA3 subcellular location (ICC-IF): Mainly localized to the nucleoplasm..
- Human Protein Atlas. FOXA3 antibody validation summary (2 antibodies).
- Transcription factor FOXA3 promotes the development of Hepatoblastoma via regulating HNF1A, AFP, and ZFHX3 expression. Journal of clinical laboratory analysis 2021 — PMC7957975.
- Opposing Roles of FoxA1 and FoxA3 in Intrahepatic Cholangiocarcinoma Progression. International journal of molecular sciences 2020 — PMC7084256.
- Molecular characterization of lung dysplasia induced by c-Raf-1. PloS one 2009 — PMC2681412.
- FOXA3 Alleviates Lipid Deposition in Primary Bovine Hepatocytes by Inhibiting SREBP1 and Cell Proliferation. Veterinary sciences 2026 — PMC12945116.
- PubMed PMID:8499623 — UniProt-cited evidence.
- PubMed PMID:10899756 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for FOXA3 (Hepatocyte nuclear factor 3-gamma). Expected localisation Nuclear staining; tissue evidence is uncertain (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet PB9805). Validated IHC antibody: PB9805 at 2-5 μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Adipose tissue (Adipocytes); negative Lymph node. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human colon cancer tissue; fixative not specified (datasheet PB9805); 2. Fixation: Image fixative and duration unreported (datasheet PB9805); 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: EDTA pH 8.0 (datasheet PB9805); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet PB9805); 8. Primary antibody: Rabbit anti-FOXA3, 2-5 μg/ml (datasheet PB9805); 9. Primary incubation: Overnight at 4 °C (datasheet PB9805). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific); Caveat: Low staining–RNA consistency warrants validation (HPA tissue IHC); Isoform / epitope: No annotated isoforms; one 1–350 chain (UniProt); Regulation: Expression regulation is unreported (UniProt). Evidence: UniProt P55318, HPA tissue IHC · Uncertain, HPA ICC-IF · Mainly localized to the nucleoplasm., Datasheet PB9805, PMC7957975, PMC7084256. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/FOXA3/immunohistochemistry-ihc-guide.