This website uses cookies to ensure you get the best experience on our website.
- Table of Contents
HNF4G / Hepatocyte nuclear factor 4-gamma · IHC design guide
Design Immunohistochemistry for HNF4G
Plan HNF4G IHC-P around nuclear staining in small-intestine endocrine cells (HPA tissue IHC). Use duodenum as a positive reference (HPA tissue IHC) and optimize the catalog antibody within 1:10–1:50 (datasheet: A09682-1).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
HNF4G 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 intestinal endocrine cells (HPA tissue IHC) | |
| Staining pattern | Strong nuclear staining in duodenal and small-intestine endocrine cells (HPA tissue IHC) | |
| Antigen retrieval | Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen) | |
| Positive control | Duodenum+2 more · see all | |
| Negative control | Adipose tissue+4 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep formalin fixation consistent across sections. (standard IHC practice; not target-specific) | |
| Caveat | Rectal endocrine staining is weaker than small-intestine staining (HPA tissue IHC) | |
| Regulation | Expression regulation not reported (UniProt) | |
| Isoform / epitope | 2 isoforms; N-terminal epitope coverage is unspecified (UniProt; datasheet: A09682-1) |
Section 1
Recommended HNF4G IHC & IF Protocols
The catalog antibody protocol is provided separately. These four published HNF4G IHC protocols provide additional conditions for lung, prostate and pancreatic cancer samples (PMC5915054; PMC5728174; PMC7862466; PMC12695649).
| Sample | FFPE human testis tissue (datasheet A09682-1) |
| Fixation | Image formalin-fixed; duration unreported (datasheet A09682-1); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat-induced epitope retrieval in Tris-EDTA buffer, pH 9.0, 20 min at 95–98 °C (standard rule: nuclear antigen) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% normal serum of the secondary host, 30 min, room temperature (standard) |
| Primary antibody | Rabbit anti-HNF4G, 1:10-1:50 (datasheet A09682-1) |
| 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 | HNF4G-positive staining in endocrine cells of duodenum (HPA tissue IHC: High). HPA tissue profile: Small intestine-specific nuclear expression. No signal in the no-primary control. |
💡Decision noteStart with Tris-EDTA pH 9.0 retrieval at 95–98 °C for 20 min (page antigen retrieval); citrate pH 6.0 is a published alternative (PMC5915054).
Section 2
What Is the Expected HNF4G Staining Pattern?
HNF4G is a nuclear transcription factor with no transmembrane segment (UniProt Q14541). In tissue IHC, expect nuclear staining in small intestinal endocrine cells, including those in duodenum (HPA: High; tissue IHC reliability Enhanced). HPA reports consistency between staining and RNA expression, with external verification pending (HPA: reliability description).
| Distinct nuclear staining in duodenal or small intestinal endocrine cells, with little surrounding stain. | This matches the reported High endocrine cell signal and small intestine-specific nuclear profile (HPA: tissue IHC). Score the fraction and intensity of stained nuclei in the relevant cells; a clear counterstain helps distinguish nuclei from adjacent cytoplasm (general IHC practice). |
| Predominantly cytoplasmic staining, a membrane rim, or stain confined to extracellular material. | That distribution does not establish the expected IHC pattern: UniProt places HNF4G in the nucleus and records no transmembrane segment (UniProt Q14541). Check morphology and detection controls before assigning such staining to HNF4G (general IHC practice). |
| Strong staining in cells outside the expected endocrine population, especially in a negative reference section. | Consider cross-reactivity or endogenous detection activity rather than calling every stained cell positive (general IHC practice). Interpret against the specific reference cell type: appendix endocrine cells were not detected, while duodenal endocrine cells were High (HPA: tissue IHC). |
| Diffuse brown haze across cells, stroma, or the entire section, without resolvable stained nuclei. | This cannot be scored as the reported nuclear pattern (HPA: tissue IHC). Background can arise from blocking, wash, reagent, or chromogen conditions; compare a no-primary control and optimize the general IHC workflow (general IHC practice). |
| No nuclear signal in a duodenal or small intestinal section expected to contain endocrine cells. | First confirm that the sampled section contains identifiable endocrine cells; HPA's High designation applies to those cells, not every cell in the tissue (HPA: tissue IHC). If present, review antibody dilution, retrieval, detection, and control performance as general IHC variables (general IHC practice). |
💡Expected HNF4G appearanceCall the IHC result positive when endocrine cell nuclei in duodenum or small intestine show distinct, relatively strong staining (HPA: High; nuclear profile); diffuse haze, membrane outlines, or staining of unrelated cells alone is an insufficient positive pattern (HPA: tissue IHC; UniProt Q14541 topology; general IHC practice).
| Reference tissue and cell type (HPA: tissue IHC) | Duodenal and small intestinal endocrine cells are reported High; rectal endocrine cells are Medium, and appendix endocrine cells are not detected (HPA: tissue IHC). Compare like cells across sections: a whole-tissue label does not mean every nucleus should stain (HPA: cell-level entries). |
| Evidence strength (HPA: tissue IHC; antibody validation) | The tissue profile is Enhanced because antibody staining is consistent with RNA expression, with external verification pending (HPA: reliability description). HPA005438 has Enhanced IHC validation; HPA010610 has no listed IHC validation status (HPA: antibody records). |
| Tissue expression versus observed staining (UniProt Q14541; HPA: tissue IHC) | UniProt lists expression in pancreas, kidney, small intestine, and testis, with weak colon expression (UniProt Q14541). The supplied HPA IHC observations specify endocrine cell staining in duodenum, small intestine, and rectum; they do not establish an IHC pattern for every UniProt-listed tissue (HPA: tissue IHC). |
| Isoforms and antibody epitope (UniProt Q14541) | Two isoforms are recorded (UniProt Q14541). No epitope location or isoform coverage is supplied, so do not infer that a staining difference identifies an isoform or that either antibody detects both (UniProt Q14541; HPA: antibody records). |
| IF/ICC Q: Should cytosolic signal be interpreted like IHC nuclear staining? | A: HPA reports nucleoplasm as the main ICC-IF location, with additional cytosol, cytokinetic bridge, and mitotic spindle locations (HPA: subcellular ICC-IF). Use that observation on the separate IF/ICC guide; the supplied tissue IHC profile remains nuclear in small intestinal endocrine cells (HPA: tissue IHC). |
| Situation | Likely cause | Next action |
|---|---|---|
| Positive tissue is uniformly blank. | The sampled level may lack recognizable endocrine cells, or an IHC workflow step may have failed (HPA: endocrine cell-specific pattern; general IHC practice). | Confirm the target cell population in the section; then check the positive control, primary antibody dilution, retrieval, and detection reagents under the chosen IHC procedure (general IHC practice). No HNF4G-specific retrieval or fixation effect is supplied. |
| Nuclei stain weakly in an expected positive section. | Rectal endocrine cells are reported Medium, whereas duodenal and small intestinal endocrine cells are High (HPA: tissue IHC); weak staining in the latter also warrants a workflow check (general IHC practice). | Record the tissue and cell type before comparing intensity. Review counterstain balance and the positive control, then optimize standard IHC conditions if the expected High cells are faint (general IHC practice). |
| Only cytoplasm or cell borders stain. | That appearance conflicts with the nuclear tissue profile and nuclear UniProt annotation (HPA: tissue IHC; UniProt Q14541); a cell-border pattern also lacks support from the recorded topology (UniProt Q14541). | Do not score it as confirmed HNF4G-positive IHC. Inspect a no-primary control, tissue morphology, and detection background; seek a reproducible nuclear pattern in the positive reference cells (general IHC practice; HPA: tissue IHC). |
| A negative reference shows convincing brown signal. | Cross-reactivity or endogenous detection activity is possible (general IHC practice); HPA reports no detectable staining in appendix endocrine cells and adipose adipocytes, among other listed cells (HPA: tissue IHC). | Match the observed cell type to the HPA negative entry, inspect no-primary and detection controls, and assess whether the signal is nuclear before changing the interpretation (HPA: tissue IHC; general IHC practice). |
| Broad diffuse stain obscures nuclear boundaries. | Nonspecific reagent binding, incomplete washing, or detection background can obscure localisation (general IHC practice); diffuse staining is not the reported small intestine-specific nuclear pattern (HPA: tissue IHC). | Compare the no-primary section, review blocking and washes, and adjust general chromogenic detection conditions until nuclei and counterstained tissue remain distinguishable (general IHC practice). |
| Two antibodies give different IHC distributions. | The supplied validation records differ: HPA005438 is Enhanced for IHC, while HPA010610 has no listed IHC status (HPA: antibody records); epitope and isoform coverage are unspecified (UniProt Q14541; HPA: antibody records). | Compare each result with the HPA nuclear endocrine cell pattern and suitable controls. Treat an unvalidated distribution as unresolved rather than attributing the discrepancy to a particular isoform (HPA: tissue IHC; general IHC practice). |
Experiment evidence
IHC & IF images showing HNF4G staining
A09682-1 · IHCFormalin-fixed and paraffin-embedded human testis tissue reacted with HNF4G Antibody (N-term), which was peroxidase-conjugated to the second
A09682 · IFImmunofluorescence analysis of U-2 OS cells using HNF4G Polyclonal Antibody at dilution of 1:100 . Blue: DAPI for nuclear staining.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000164749-HNF4G/tissue/cerebral+cortex#imid_19768161
HPA · DuodenumHuman Protein Atlas IHC, Duodenum. Source: https://www.proteinatlas.org/ENSG00000164749-HNF4G/tissue/duodenum#imid_19770830
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000164749-HNF4G/tissue/colon#imid_19770815
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000164749-HNF4G/tissue/liver#imid_19770824
HPA · ICC-IF A-549Human Protein Atlas immunofluorescence, A-549, antibody HPA010610; green = HNF4G, blue = nucleus, red = microtubules.
HPA · ICC-IF A-549Human Protein Atlas immunofluorescence, A-549, antibody HPA010610; green = HNF4G, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for HNF4G IHC & IF
🧪Run duodenum first and score its endocrine cells for nuclear staining (High; HPA: duodenum endocrine cells; UniProt Q14541: nucleus). Use appendix endocrine cells as a negative tissue (Not detected; HPA: appendix endocrine cells); on the duodenum slide, surrounding cells should provide an internal check for nonspecific staining, without assuming every nonendocrine cell is negative (HPA: duodenum endocrine cells High).
Positive control tissue: Duodenum (Endocrine cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show HNF4G in A-549, PC-3, U2OS, with annotated localisation: Nucleoplasm (enhanced) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control and an isotype control matched to the primary antibody’s host species, isotype and concentration; confirm target specificity with knockout tissue or a peptide block when the relevant material is available (standard IHC practice). For chromogenic detection, block endogenous peroxidase and check endogenous biotin if using a biotin-based system (standard IHC practice).
⚠️Feasibility: The selected A09682-1 testis IHC caption explicitly reports formalin-fixed, paraffin-embedded tissue, but supplies no HNF4G-specific fixation window or fixation effect (A09682-1 tissue-IHC caption). Retrieval dependence is unreported; assess antigen retrieval with the catalog antibody’s IHC-P protocol as the starting point (supplied application evidence: catalog IHC-P protocol). The supplied evidence does not establish that frozen sections or IF are easier; intestinal sections warrant attention to endogenous peroxidase background during chromogenic detection (standard IHC practice).
HPA tissue IHC evidence for HNF4G
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — High 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 |
|---|---|---|---|---|
| Duodenum | Endocrine cells | High | Protein (IHC) | HPA → |
| Small intestine | Endocrine cells | High | Protein (IHC) | HPA → |
| Rectum | Endocrine 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 → |
| Appendix | Endocrine cells | Not detected | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Breast | Adipocytes | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced HNF4G IHC Tips
Troubleshoot HNF4G staining by checking nuclear signal in expected cells, then evaluating retrieval, detection background and scoring consistency (UniProt Q14541; HPA tissue IHC).
How should I retrieve HNF4G in paraffin sections when nuclear staining is weak?
Use Tris-EDTA at pH 9.0 for heat-induced retrieval at 95–98 °C for 20 minutes before staining HNF4G (page retrieval setting). Allow sections to cool in the retrieval buffer, then rinse consistently across the run so differences in handling do not confound nuclear staining (standard IHC practice). Include a small-intestine control with expected endocrine-cell nuclear signal while optimizing the catalog antibody (HPA tissue IHC: high in small-intestine endocrine cells; UniProt Q14541: nucleus). If signal remains weak, compare a second retrieval condition on adjacent sections while holding antibody concentration, detection and imaging constant (standard IHC practice).
Could fixation explain variable HNF4G staining across paraffin blocks?
Target-specific sensitivity of HNF4G to fixation duration or fixative chemistry is unknown from the supplied evidence; treat block-to-block differences as a testable possibility (supplied HNF4G record). Compare sections with documented processing histories using the same pH 9.0 retrieval and the same staining run (page retrieval setting; standard IHC practice). Assess nuclear signal in the expected cell population alongside overall morphology, since poorly preserved nuclei make chromogenic scoring unreliable (HPA tissue IHC: small-intestine endocrine cells; standard IHC practice). If only one block fails, repeat staining with a suitable positive-control section before attributing the difference to fixation (standard IHC practice).
What should I do when HNF4G appears predominantly cytoplasmic in IHC?
For chromogenic tissue scoring, prioritize a clear nuclear pattern: HNF4G is annotated as nuclear, and tissue IHC describes small-intestine-specific nuclear expression (UniProt Q14541; HPA tissue IHC). HPA cell imaging also reports nucleoplasmic signal with additional cytosolic localization, so isolated cytoplasmic color should trigger review rather than automatic rejection (HPA subcellular). Compare the suspect section with a positive-control section in the same staining run and inspect whether the nuclear counterstain permits confident compartment assignment (standard IHC practice). If cytoplasmic DAB is widespread across unrelated cells, check secondary-only staining and endogenous peroxidase blocking before scoring it as HNF4G (standard IHC practice).
How can isoforms or epitope accessibility affect HNF4G IHC?
HNF4G has 2 annotated isoforms, and its nuclear receptor ligand-binding domain spans residues 99–328 (UniProt Q14541). The selected tissue-IHC caption identifies an N-terminal antibody, but it does not establish which isoforms its epitope recognizes (selected A09682-1 tissue-IHC caption; UniProt Q14541). Confirm the exact immunogen or epitope information for the antibody being used before interpreting discordant staining across samples (standard antibody-validation practice). HNF4G has annotated phosphorylation sites at residues 94, 370, 373 and 377; whether these alter this antibody’s binding in fixed sections is unknown (UniProt Q14541). Compare retrieval conditions on adjacent sections if epitope masking remains a concern (standard IHC practice).
How should I adapt this HNF4G assessment for multiplex IF?
For the separate IF workflow, pair HNF4G with a validated marker of the expected endocrine-cell population and include a nuclear counterstain (HPA tissue IHC: small-intestine endocrine cells; standard IF practice). Choose fluorophores after checking unstained tissue autofluorescence, and place the weaker expected signal in a cleaner spectral channel (standard IF practice). HNF4G is nuclear and has no annotated transmembrane segment, so use permeabilisation appropriate for access to an intracellular nuclear epitope (UniProt Q14541; standard IF practice). Optimize permeabilisation against morphology and background, then use single-stain controls to assess channel bleed-through before interpreting nuclear overlap (standard IF practice).
How do I distinguish diffuse DAB background from weak HNF4G staining?
Expected tissue staining is nuclear in a restricted cell population, with high signal reported in small-intestine endocrine cells (HPA tissue IHC). If DAB appears throughout the section, inspect a no-primary control and verify the peroxidase block before changing the primary-antibody conditions (standard chromogenic IHC practice). Check whether precipitate follows section folds, drying edges or debris rather than intact nuclei; those patterns can create misleading brown deposits (standard IHC practice). Compare background and nuclear contrast at the same microscope settings, then adjust blocking, washes or detection development one variable at a time (standard IHC practice). Retain a positive-control section in each comparison (standard IHC practice).
What is a defensible way to quantify HNF4G-positive cells in tissue? ⚠ ANSWER MARKED FOR VERIFICATION
Define the region and cell population before scoring, because HPA reports high expression in small-intestine endocrine cells rather than uniform staining across all cells (HPA tissue IHC). Score nuclear signal using % positive cells or an H-score based on intensity and proportion, with one threshold applied across comparable sections (standard IHC practice). If cell abundance itself is the question, report positive-cell density per mm² of viable tissue (standard histologic quantification practice). Normalize positive nuclei to all evaluable nuclei in the defined population, or density to measured viable area; record exclusions for folds and necrosis (standard IHC practice).
When is a brown nuclear signal convincing evidence of HNF4G?
A convincing result places nuclear signal in an expected population, such as small-intestine endocrine cells, and shows a comparable pattern in the run’s positive control (HPA tissue IHC; standard IHC practice). The selected antibody has a formalin-fixed, paraffin-embedded human testis DAB image, which demonstrates an IHC application but does not establish clinical relevance (selected A09682-1 tissue-IHC caption). Treat dominant staining in unrelated cells or only outside nuclei as a prompt to reassess specificity and detection background (UniProt Q14541: nucleus; standard IHC practice). Exclude edge effects and necrotic areas, and use a no-primary control to investigate endogenous enzyme signal or nonspecific DAB deposition (standard IHC practice).
Boster reagents
Best HNF4G / Hepatocyte nuclear factor 4-gamma IHC Antibodies
The catalog pairs an IHC-P antibody with a chromogenic image of formalin-fixed, paraffin-embedded human testis and an IF/ICC antibody with an image of U-2 OS cells (catalog: applications; image captions).
Real IHC data
Anti-HNF4G Antibody (N-term)
Cat # A09682-1
Real IF data
Anti-HNF4G Antibody
Cat # A09682
A09682-1 is listed for human IHC-P, with DAB staining shown in formalin-fixed, paraffin-embedded human testis (catalog: applications and reactivity; IHC image caption). A09682 is listed for IF/ICC in Human, Mouse and Rat, with IF shown in U-2 OS cells (catalog: applications and reactivity; IF image caption).
Which to pick: Choose A09682-1 for human tissue IHC-P: its own image documents formalin-fixed, paraffin-embedded testis stained with DAB, and its listed dilution is 1:10–1:50 (IHC image caption; datasheet: IHC-P dilution). Choose A09682 for IF/ICC: its own image shows U-2 OS cells with DAPI nuclear staining, and its listed dilution is 1:50–1:100 (IF image caption; datasheet: ICC/IF dilution). For Mouse or Rat samples, A09682 has listed reactivity and a polyclonal format, but its supplied IF image documents U-2 OS cells only (catalog: reactivity and clonality; IF image caption).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q14541 (HNF4G_HUMAN, Hepatocyte nuclear factor 4-gamma).
- Human Protein Atlas. HNF4G tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. HNF4G subcellular location (ICC-IF): Mainly localized to the nucleoplasm. In addition localized to the cytosol, cytokinetic bridge and mitotic spindle..
- Human Protein Atlas. HNF4G antibody validation summary (2 antibodies).
- Expression of HNF4G and its potential functions in lung cancer. Oncotarget 2018 — PMC5915054.
- Aberrant Activation of a Gastrointestinal Transcriptional Circuit in Prostate Cancer Mediates Castration Resistance. Cancer cell 2017 — PMC5728174.
- Metformin inhibits pancreatic cancer metastasis caused by SMAD4 deficiency and consequent HNF4G upregulation. Protein & cell 2021 — PMC7862466.
- Transcription factor switching drives subtype-specific pancreatic cancer. Nature genetics 2025 — PMC12695649.
- PubMed PMID:8622695 — UniProt-cited evidence.
- PubMed PMID:17974005 — UniProt-cited evidence.
- PubMed PMID:10512380 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for HNF4G (Hepatocyte nuclear factor 4-gamma). Expected localisation Nuclear staining in intestinal endocrine cells (HPA tissue IHC). Antigen retrieval: Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen). Validated IHC antibody: A09682-1 at 1:10-1:50 (the product's own IHC validation image is shown). Controls (HPA): positive Duodenum (Endocrine cells); negative Adipose tissue. IHC-P workflow on FFPE sections. Protocol steps: 1. Sample: FFPE human testis tissue (datasheet A09682-1); 2. Fixation: Image formalin-fixed; duration unreported (datasheet A09682-1); 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-induced epitope retrieval in Tris-EDTA buffer, pH 9.0 , 20 min at 95–98 °C…; 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% normal serum of the secondary host, 30 min , room temperature (standard); 8. Primary antibody: Rabbit anti-HNF4G, 1:10-1:50 (datasheet A09682-1); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep formalin fixation consistent across sections. (standard IHC practice; not target-specific); Caveat: Rectal endocrine staining is weaker than small-intestine staining (HPA tissue IHC); Isoform / epitope: 2 isoforms; N-terminal epitope coverage is unspecified (UniProt; datasheet: A09682-1); Regulation: Expression regulation not reported (UniProt). Evidence: UniProt Q14541, HPA tissue IHC · Enhanced, HPA ICC-IF · Mainly localized to the nucleoplasm. In addition localized to the…, Datasheet A09682-1, PMC5915054, PMC5728174. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/HNF4G/immunohistochemistry-ihc-guide.