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HOXA4 / Homeobox protein Hox-A4 · IHC design guide
Design Immunohistochemistry for HOXA4
Plan HOXA4 paraffin-section IHC around general nuclear staining (HPA tissue IHC) and a catalog antibody dilution of 0.5–1 μg/mL (datasheet: PA1603 IHC-P). Compare high-staining glandular or neuronal cells with parathyroid glandular cells reported as undetected, while accounting for low staining–RNA concordance (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
HOXA4 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 | General nuclear staining (HPA tissue IHC) | |
| Staining pattern | Nuclear signal in glandular and neuronal cells (HPA tissue IHC) | |
| Antigen retrieval | Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen) | |
| Positive control | Adrenal gland+4 more · see all | |
| Negative control | Parathyroid gland |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image PA1603) | |
| Caveat | Staining and RNA levels have low concordance (HPA tissue IHC) | |
| Regulation | Embryonic nervous system expression (UniProt) | |
| Isoform / epitope | No isoforms or processing variants annotated (UniProt) |
Section 1
Recommended HOXA4 IHC & IF Protocols
The catalog antibody’s IHC-P protocol is accompanied by three published HOXA4 IHC protocols (PMC3125234; PMC4750173; PMC9859321).
| Sample | Tissue sections; selected-image fixative not specified (standard IHC workflow) |
| Fixation | Image fixative and duration unreported (datasheet PA1603); 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-HOXA4, 0.5-1μg/ml (datasheet PA1603) |
| 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 | HOXA4-positive staining in glandular cells of adrenal gland (HPA tissue IHC: High). HPA tissue profile: General 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 retrieval setting; UniProt: nucleus).
Section 2
What Is the Expected HOXA4 Staining Pattern?
HOXA4 is a nuclear transcription factor with no transmembrane segment (UniProt Q00056). In tissue IHC, HPA reports general nuclear staining, including high staining in selected glandular, neuronal, and squamous epithelial cells (HPA tissue IHC). HPA rates the tissue staining Approved but reports low consistency with RNA expression and pending external verification (HPA tissue IHC). Interpret a positive stain in its tissue and cell context.
| Distinct nuclear staining in the expected cells. | This matches the reported compartment (UniProt Q00056; HPA tissue IHC). High staining in cerebellar Purkinje cells or colon glandular cells is an observed reference pattern (HPA tissue IHC). Compare nuclear signal with neighboring cells on the same section; intensity alone does not establish specificity (general IHC practice). |
| Predominantly cytoplasmic or membranous staining. | That distribution conflicts with the reported nuclear localization and absence of a transmembrane segment (UniProt Q00056). Treat it as suspect; review morphology, detection background, and an appropriate control before scoring it as HOXA4 (general IHC practice). |
| Strong signal in a cell population outside the expected pattern. | HPA's listed high staining applies to particular cells within each tissue, not every cell in that tissue (HPA tissue IHC). Cross-reactivity or endogenous detection activity are possible explanations (general IHC practice); check the cell identity and controls before assigning the signal to HOXA4. |
| Diffuse color across nuclei, cytoplasm, and surrounding tissue. | This does not resolve the reported nuclear pattern (HPA tissue IHC). Background from detection reagents, inadequate blocking, or excessive antibody concentration can obscure localization (general IHC practice). Judge the stain against a reagent control processed with the section. |
| No nuclear signal in a listed high-staining tissue. | An absent signal in the relevant cell population warrants a technical check, although HPA's Approved rating has an RNA-consistency caveat (HPA tissue IHC). Confirm the cells are present, then review controls and the catalog antibody's IHC-P instructions (general IHC practice). |
💡Expected HOXA4 appearanceCall a result consistent with HOXA4 when chromogenic signal is predominantly nuclear and strong in a listed high-staining cell population, such as cerebellar Purkinje cells (HPA tissue IHC; UniProt Q00056); broad cytoplasmic or membranous color is suspect (UniProt Q00056; general IHC practice).
| Tissue and cell selection | HPA reports high staining in adrenal and appendix glandular cells, cerebellar Purkinje cells, cerebral cortex neuronal cells, and esophageal squamous epithelial cells (HPA tissue IHC). Its parathyroid glandular cells are listed as not detected (HPA tissue IHC). Use the stated cell population when comparing sections. |
| Strength of the tissue evidence | The tissue profile is Approved, with low consistency between antibody staining and RNA expression and external verification pending (HPA tissue IHC). The listed staining is an observed reference, not proof that every positive cell contains HOXA4. HPA lists HPA060088 as IHC Approved; the payload does not report IHC Enhanced validation (HPA antibodies). |
| Compartment and topology | UniProt places HOXA4 in the nucleus and lists no transmembrane segment (UniProt Q00056). HPA describes general nuclear tissue staining (HPA tissue IHC). Score the nuclear compartment first; a strong signal elsewhere needs independent scrutiny before interpretation (general IHC practice). |
| Processing and annotated forms | UniProt lists one chain spanning residues 1–320, no signal peptide or propeptide, no annotated isoforms, and no glycosylation sites (UniProt Q00056). These annotations provide no basis for predicting a secreted or membrane staining pattern; they do not predict formalin sensitivity or retrieval requirements. |
| IF/ICC: what pattern is reported? | HPA reports HOXA4 in nuclear bodies in ICC-IF, with images from HEK293, Hep-G2, and U2OS (HPA subcellular). This supports a punctate nuclear pattern in that application. It does not establish an IHC-P staining intensity or an IF/ICC protocol. |
| Situation | Likely cause | Next action |
|---|---|---|
| Expected nuclei appear unstained in a listed high-staining tissue. | The relevant cells may be absent from the field, or the staining run may have failed (general IHC practice); HPA's tissue result also has an RNA-consistency caveat (HPA tissue IHC). | Identify the specified cells on the counterstain, check a run control, and compare the procedure with the catalog antibody's IHC-P instructions (general IHC practice). Do not infer a HOXA4-specific fixation effect from this result. |
| The section stains mainly at cell borders or in cytoplasm. | That compartment disagrees with nuclear HOXA4 localization and its lack of a transmembrane segment (UniProt Q00056); nonspecific staining is possible (general IHC practice). | Check a reagent control and the detection system, then rescore only morphologically identifiable nuclei (general IHC practice). Do not count border staining as a HOXA4-positive cell. |
| Many unrelated cell types show equally strong color. | The pattern lacks the cell specificity needed to compare with HPA's cell-level observations (HPA tissue IHC). Cross-reactivity or endogenous detection activity may contribute (general IHC practice). | Compare the named cell population with adjacent cells; check a control omitting primary antibody and assess endogenous detection activity as appropriate to the chromogen system (general IHC practice). |
| Hematoxylin obscures a weak nuclear signal. | A heavy counterstain can make low chromogenic signal difficult to judge (general IHC practice). HPA also lists low staining in hippocampal neuronal cells and pancreatic exocrine glandular cells (HPA tissue IHC). | Review counterstain balance and use a listed high-staining cell population to assess the run before calling a low-staining population negative (general IHC practice; HPA tissue IHC). |
| Diffuse background prevents compartment scoring. | Excess detection signal, insufficient blocking, or concentrated primary antibody can raise background (general IHC practice); the supplied sources give no HOXA4-specific background mechanism. | Check reagent controls, blocking, washes, and the dilution specified for the IHC-validated antibody (general IHC practice). Score only when nuclei can be distinguished from surrounding tissue. |
| Parathyroid glandular cells stain strongly. | HPA lists this population as not detected (HPA tissue IHC), but one discordant section does not establish whether the signal is biological or technical (general IHC practice). | Confirm cell identity, examine a reagent control, and compare a listed high-staining population in the same run (general IHC practice; HPA tissue IHC). Report the discrepancy rather than treating the HPA negative call as an absolute exclusion. |
Experiment evidence
IHC & IF images showing HOXA4 staining
PA1603 · IHCAnti-HOXA4 antibody, PA1603, IHC(P) IHC(P): Human Mammary Cancer Tissue
PA1603 · IFAnti-HOXA4 antibody, PA1603, ICC ICC: HELA Cell
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000197576-HOXA4/tissue/cerebral+cortex#imid_16485123
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000197576-HOXA4/tissue/colon#imid_16487793
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000197576-HOXA4/tissue/liver#imid_16487833
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000197576-HOXA4/tissue/kidney#imid_16487877
HPA · ICC-IF HEK293Human Protein Atlas immunofluorescence, HEK293, antibody HPA060088; green = HOXA4, blue = nucleus, red = microtubules.
HPA · ICC-IF HEK293Human Protein Atlas immunofluorescence, HEK293, antibody HPA060088; green = HOXA4, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for HOXA4 IHC & IF
🧪Run appendix first and score nuclear staining in its glandular cells (HPA: High in appendix glandular cells; UniProt Q00056: nucleus). Run parathyroid gland as the negative tissue (HPA: Not detected in parathyroid glandular cells); on the appendix slide, use surrounding cells without nuclear signal as an internal background reference, rather than assuming every nonglandular cell is HOXA4-negative (standard IHC practice).
Positive control tissue: Adrenal gland (Glandular cells, HPA High)
Negative control tissue: Parathyroid gland (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show HOXA4 in HEK293, Hep-G2, U2OS, with annotated localisation: Nuclear bodies (supported) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control and a control matched to the primary antibody’s host species and antibody class, with an isotype match for a monoclonal primary (standard IHC practice). Use HOXA4-knockout material as a biological negative where available, or validated cognate-peptide competition (standard IHC practice).
Check for endogenous peroxidase signal in appendix blood and immune cells before interpreting chromogenic staining (standard IHC practice).
Check for endogenous peroxidase signal in appendix blood and immune cells before interpreting chromogenic staining (standard IHC practice).
⚠️Feasibility: A target-specific fixation window and retrieval dependency are unreported in the supplied evidence; establish retrieval conditions empirically for paraffin sections (standard IHC practice). The PA1603 tissue-IHC caption shows human mammary cancer tissue but does not report its fixative (selected PA1603 IHC(P) caption). The supplied evidence does not establish whether frozen sections or IF are easier for HOXA4; in IF, check nuclear-body localization against the reported pattern and assess tissue autofluorescence (HPA: nuclear bodies supported; standard IF practice).
HPA tissue IHC evidence for HOXA4
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — 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 |
|---|---|---|---|---|
| Adrenal gland | Glandular cells | High | Protein (IHC) | HPA → |
| Appendix | Glandular cells | High | Protein (IHC) | HPA → |
| Cerebellum | Purkinje cells | High | Protein (IHC) | HPA → |
| Cerebral cortex | Neuronal cells | High | Protein (IHC) | HPA → |
| Cervix | Glandular cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Parathyroid gland | Glandular cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced HOXA4 IHC Tips
Troubleshoot HOXA4 staining by checking nuclear localisation, section handling and assay controls before interpreting staining intensity (UniProt Q00056; HPA tissue IHC).
How should I adjust retrieval when HOXA4 nuclear staining is weak?
Start with Tris-EDTA, pH 9.0, heat-induced retrieval at 95–98 °C for 20 min (page retrieval rule: nuclear antigen). Run a known positive section alongside the test section, then compare nuclear signal and tissue integrity at the same detection settings (HPA tissue IHC: general nuclear expression; standard IHC practice). If staining remains weak, test a shorter heating period or citrate, pH 6.0, on serial sections as fallback conditions (standard IHC practice). Change one variable at a time and reject conditions that increase diffuse cytoplasmic colour without improving interpretable nuclear staining (UniProt Q00056: nucleus; standard IHC practice).
Could fixation explain weak or uneven HOXA4 staining in paraffin sections?
HOXA4-specific sensitivity to fixation is unknown from the supplied evidence, and the PA1603 tissue caption does not state a fixative (PA1603 IHC(P) caption). Record the fixative and fixation duration for each specimen, and compare sections of similar thickness processed together (standard IHC practice). For a controlled troubleshooting run, hold Tris-EDTA retrieval at pH 9.0, 95–98 °C for 20 min while varying only the available fixation condition (page retrieval rule: nuclear antigen; standard IHC practice). Assess preserved morphology and nuclear staining together; neither HPA staining patterns nor HOXA4 topology establishes a target-specific fixation effect (HPA tissue IHC; UniProt Q00056; standard IHC practice).
How should I judge cytoplasmic colour in a HOXA4 IHC section?
Score nuclear staining as the primary HOXA4 signal because UniProt places this transcription factor in the nucleus (UniProt Q00056: subcellular location and function). HPA describes general nuclear tissue expression, while its cell imaging supports a nuclear-body location; these observations guide compartment checks but do not validate every chromogenic pattern (HPA tissue IHC; HPA subcellular). Compare the suspect section with a positive section and a no-primary control under the same detection conditions (standard IHC practice). If cytoplasmic colour dominates, inspect retrieval damage, diffuse background and endogenous peroxidase before assigning it biological meaning (standard IHC practice). Report the nuclear result separately from unexplained cytoplasmic staining (UniProt Q00056: nucleus; standard IHC practice).
Can an unreported HOXA4 epitope explain discordant nuclear staining?
The supplied record lists one chain spanning residues 1–320 and no annotated isoforms, glycosylation sites or modified residues (UniProt Q00056: processing, isoforms and modifications). Those annotations do not identify the catalog antibody’s epitope or establish its accessibility after processing (UniProt Q00056; PA1603 IHC(P) caption). Request the immunogen or epitope specification before attributing discordant sections to epitope masking (standard IHC practice). Compare serial sections with the same retrieval and detection settings, and use an independently validated antibody if epitope-dependent staining remains a concern (standard IHC practice). Treat a changed pattern as provisional until nuclear localisation and appropriate controls agree (UniProt Q00056: nucleus; standard IHC practice).
How can IF help check a puzzling HOXA4 IHC pattern?
Use IF on a matched section as a separate assay, and verify that the chosen antibody works in IF before comparing it with chromogenic IHC (standard IF practice; PA1603 IHC(P) caption). Multiplex HOXA4 with a marker for the expected cell population and a nuclear counterstain, then inspect whether signal occupies those nuclei (HPA tissue IHC: cell-specific patterns; UniProt Q00056: nucleus; standard IF practice). Choose fluorophores after measuring tissue autofluorescence, favouring a spectral channel with clear separation from that background (standard IF practice). Permeabilise sufficiently for an antibody to reach the nuclear epitope; HOXA4 has no transmembrane segment (UniProt Q00056: topology and location; standard IF practice). HPA nuclear-body IF observations provide a localisation reference, not validation of the catalog antibody for IF (HPA subcellular; PA1603 IHC(P) caption).
What controls distinguish HOXA4 signal from brown background?
Run a no-primary control through the same chromogenic detection steps to locate reagent-dependent colour (standard IHC practice). Apply a peroxidase block before horseradish-peroxidase detection, and compare DAB development times across matched sections (standard IHC practice). Inspect folds, section edges, pigment and damaged areas separately from intact nuclei because these features can produce misleading colour (standard IHC practice). If background rises with stronger retrieval, return to Tris-EDTA, pH 9.0, 95–98 °C for 20 min and optimise detection on serial sections (page retrieval rule: nuclear antigen; standard IHC practice). Accept a result only when nuclear staining is distinguishable from the no-primary control and preserved tissue background (UniProt Q00056: nucleus; standard IHC practice).
How should I quantify heterogeneous HOXA4 nuclear staining? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and analysis regions before scoring, and exclude folds, edges and necrotic areas by a consistent rule (standard IHC practice). Record the percentage of positive nuclei and intensity categories, then calculate a nuclear H-score on the 0–300 scale if intensity is reproducible (standard IHC practice; UniProt Q00056: nucleus). Alternatively, report positive nuclei per mm² when cell density varies across sampled regions (standard IHC practice). Normalise counts to all evaluable nuclei in the same defined cell population, or to the measured viable tissue area for density estimates (standard IHC practice). Keep retrieval, detection, thresholds and sampling rules constant across compared sections (standard IHC practice).
When is a HOXA4-positive IHC result convincing?
Look for nuclear colour in intact cells that exceeds the matched no-primary control, with interpretable morphology across the section (UniProt Q00056: nucleus; standard IHC practice). HPA reports high staining in several glandular and neuronal cell populations, but also flags low consistency between antibody staining and RNA data and pending external verification (HPA tissue IHC: profile and reliability). Check that the stained cell population fits the sampled tissue before treating intensity as HOXA4 expression (HPA tissue IHC; standard IHC practice). Discount edge accentuation, necrotic regions, predominantly cytoplasmic colour and colour persisting after primary omission (UniProt Q00056: nucleus; standard IHC practice). Resolve unexpected patterns with an independent antibody or orthogonal expression evidence before making a biological claim (HPA tissue IHC: reliability; standard IHC practice).
Boster reagents
Best HOXA4 / Homeobox protein Hox-A4 IHC Antibodies
PA1603 has IHC image data from human mammary cancer tissue and ICC image data from HeLa cells (catalog image captions); its listed reactivity is human, mouse and rat (catalog: reactivity).
Real IHC data
Anti-Homeobox protein Hox-A4 HOXA4 Antibody ®
Cat # PA1603
PA1603 is listed for IHC and ICC, with an IHC(P) image of human mammary cancer tissue (catalog: applications; PA1603 IHC image caption). Its ICC image shows HeLa cells, and its listed reactivity includes human, mouse and rat (PA1603 ICC image caption; catalog: reactivity).
Which to pick: Choose PA1603 for paraffin-section IHC: its tissue image is labeled IHC(P), and its IHC dilution is 0.5–1 μg/ml (PA1603 IHC image caption; datasheet: IHC dilution). For ICC, PA1603 has a HeLa cell image and a listed dilution of 0.5–1 μg/ml; IF is not separately listed as an application (PA1603 ICC image caption; datasheet: ICC dilution; catalog: applications). PA1603 also lists mouse and rat reactivity, but its IHC image documents human tissue only; the caption does not report a fixative, and clonality is unreported (catalog: reactivity; PA1603 IHC image caption; catalog: clone).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q00056 (HXA4_HUMAN, Homeobox protein Hox-A4).
- Human Protein Atlas. HOXA4 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. HOXA4 subcellular location (ICC-IF): Localized to the nuclear bodies..
- Human Protein Atlas. HOXA4 antibody validation summary (1 antibodies).
- Regional expression of HOXA4 along the aorta and its potential role in human abdominal aortic aneurysms. BMC physiology 2011 — PMC3125234.
- HOX transcription factors are potential targets and markers in malignant mesothelioma. BMC cancer 2016 — PMC4750173.
- Changes in Hox Gene Chromatin Organization during Odontogenic Lineage Specification. Genes 2023 — PMC9859321.
- Homeobox A4 suppresses vascular remodeling by repressing YAP/TEAD transcriptional activity. EMBO reports 2020 — PMC7132199.
- PubMed PMID:1675427 — UniProt-cited evidence.
- PubMed PMID:1981366 — UniProt-cited evidence.
- PubMed PMID:12853948 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for HOXA4 (Homeobox protein Hox-A4). Expected localisation General nuclear staining (HPA tissue IHC). Antigen retrieval: Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen). Validated IHC antibody: PA1603 at 0.5-1μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Adrenal gland (Glandular cells); negative Parathyroid gland. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Tissue sections; selected-image fixative not specified (standard IHC workflow); 2. Fixation: Image fixative and duration unreported (datasheet PA1603); 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-HOXA4, 0.5-1μg/ml (datasheet PA1603); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image PA1603); Caveat: Staining and RNA levels have low concordance (HPA tissue IHC); Isoform / epitope: No isoforms or processing variants annotated (UniProt); Regulation: Embryonic nervous system expression (UniProt). Evidence: UniProt Q00056, HPA tissue IHC · Approved, HPA ICC-IF · nuclear bodies., Datasheet PA1603, PMC3125234, PMC4750173. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/HOXA4/immunohistochemistry-ihc-guide.