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PHOX2A / Paired mesoderm homeobox protein 2A · IHC design guide
Design Immunohistochemistry for PHOX2A
Plan PHOX2A chromogenic IHC around high staining in adrenal medullary cells (HPA tissue IHC) and expected nuclear localisation (UniProt). Use the catalog antibody’s 2.5 μg/mL starting concentration (datasheet) and compare staining with HPA tissue controls (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
PHOX2A 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 localisation is expected; tissue staining compartment is unconfirmed (UniProt) | |
| Staining pattern | High staining in adrenal medullary cells; compartment unreported (HPA tissue IHC) | |
| Antigen retrieval | Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen) | |
| Positive control | Adrenal gland+1 more · see all | |
| Negative control | Adipose tissue+4 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent across sections (standard IHC practice; not target-specific) | |
| Caveat | A section lacking adrenal medullary cells may miss the positive population (HPA tissue IHC) | |
| Regulation | Adrenal gland–enriched expression (HPA tissue RNA) | |
| Isoform / epitope | No annotated isoforms or processing; epitope effects unknown (UniProt) |
Section 1
Recommended PHOX2A IHC & IF Protocols
The catalog antibody provides the IHC-P protocol; the published PHOX2A immunohistochemistry protocol below uses mouse and human tissue with fluorescent detection.
| Sample | Tissue sections; selected-image fixative not specified (standard IHC workflow) |
| Fixation | Image fixative and duration unreported (datasheet A05780); 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-PHOX2A, 2.5 μg/mL (datasheet A05780) |
| 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 | PHOX2A-positive staining in medullary cells of adrenal gland (HPA tissue IHC: High). HPA tissue profile: Expression in adrenal medulla. No signal in the no-primary control. |
💡Decision noteFor paraffin sections, start with Tris-EDTA pH 9.0 retrieval at 95–98 °C for 20 min (page retrieval rule: nuclear antigen).
Section 2
What Is the Expected PHOX2A Staining Pattern?
PHOX2A is a nuclear protein with no transmembrane segment (UniProt O14813 localization and topology). In paraffin section IHC, expect staining in adrenal medullary cells, where HPA reports high expression, and potentially in preleptotene spermatocytes, where it reports medium expression (HPA tissue IHC). HPA rates the tissue pattern Enhanced because antibody staining agrees strongly with RNA expression (HPA tissue IHC reliability).
| Distinct nuclear staining in adrenal medullary cells; neighboring cells show little or no signal. | This matches the principal observed tissue pattern: high staining in medullary cells and adrenal enriched RNA expression (HPA tissue IHC). Judge the result by cell identity and nuclear placement together; staining somewhere in the adrenal gland alone is less informative (UniProt O14813 localization; HPA tissue IHC). |
| Nuclear staining in preleptotene spermatocytes is weaker than the adrenal medullary signal. | A medium testicular signal is compatible with the reported pattern (HPA tissue IHC: Medium in preleptotene spermatocytes; High in adrenal medullary cells). Do not require testis to match adrenal intensity, or treat all testicular cell types as established positives. |
| The strongest staining fills cytoplasm or outlines cell membranes while nuclei remain clear. | That distribution conflicts with the nuclear assignment and lack of a transmembrane segment (UniProt O14813 localization and topology). Consider nonspecific staining or a detection artefact, then reassess nuclear detail and controls before assigning PHOX2A positivity (general IHC practice). |
| Strong chromogen appears in cell types listed as undetected, or across many unrelated cells. | For example, HPA reports no detection in adipocytes of adipose tissue or hematopoietic cells of bone marrow (HPA tissue IHC). Broad staining there raises concern for cross-reactivity or endogenous detection activity; those HPA observations do not establish that every cell in either tissue must be negative. |
| No nuclear signal appears in identifiable adrenal medullary cells. | The result misses the strongest supplied positive reference (HPA tissue IHC: High in adrenal medullary cells). Check section identity, antibody and detection controls, and the validated staining conditions before interpreting the slide as PHOX2A negative (general IHC practice). |
💡Expected PHOX2A appearanceCall a convincing positive result when adrenal medullary cells show clear, predominantly nuclear chromogen with strong relative intensity (HPA tissue IHC: High; UniProt O14813 localization); broad cytoplasmic or membrane staining without nuclear definition is suspect (UniProt O14813 localization and topology).
| Cell identity and tissue context | Adrenal medullary cells are the strongest supplied tissue reference; preleptotene spermatocytes have medium staining (HPA tissue IHC). Use the named cells when comparing slides: the HPA levels describe observed cells, not a blanket score for every cell in those organs. |
| Antibody evidence for IHC | HPA rates the tissue IHC profile Enhanced and lists HPA078004 as IHC Enhanced (HPA tissue IHC; HPA antibodies). Those ratings support the reported pattern; they do not establish the performance of every other antibody or every staining condition. |
| Protein location and processing | PHOX2A is nuclear, with no transmembrane segment, signal peptide, propeptide, or annotated processed fragment (UniProt O14813). Read a discrete nuclear result as the expected compartment; the supplied record gives no basis for expecting a membrane or secreted staining pattern. |
| IF/ICC Q: where should fluorescence appear? | A: Mainly in the nucleoplasm (HPA subcellular ICC-IF: supported). HPA lists SH-SY5Y and U2OS images and marks HPA053810 and HPA065621 ICC Approved (HPA subcellular; HPA antibodies). This supports an IF localization comparison, not an IHC-P protocol. |
| Fixation and retrieval evidence | Target-specific fixation effects are not established by the supplied assay evidence. Verify with a matched IHC source before attributing a result to fixation. |
| Situation | Likely cause | Next action |
|---|---|---|
| Adrenal medulla has no visible nuclear staining. | A missed positive tissue pattern may reflect a staining or detection failure; HPA reports high medullary cell staining (HPA tissue IHC). | Confirm medullary cells are present, inspect a working positive control, and check primary antibody, retrieval, and detection steps against the IHC-validated antibody's instructions (general IHC practice). |
| Nuclei are clear but cytoplasm is strongly colored. | The compartment conflicts with PHOX2A's nuclear location (UniProt O14813). Nonspecific binding or detection background is possible (general IHC practice). | Compare with a primary-omission control; review blocking, antibody concentration, wash stringency, and counterstain visibility (general IHC practice). Require a cell-specific nuclear pattern for interpretation. |
| Many cell types show uniform chromogen, including cells expected to be undetected. | Widespread staining conflicts with the restricted HPA observations and may reflect cross-reactivity or endogenous detection activity (HPA tissue IHC; general IHC practice). | Inspect a primary-omission control and the detection-system blocking steps; compare the same run with adrenal medulla and an HPA-listed undetected cell type (general IHC practice; HPA tissue IHC). |
| All sections show diffuse haze that obscures nuclei. | Excess antibody or incomplete blocking or washing can raise background in chromogenic IHC (general IHC practice). The supplied sources do not define a PHOX2A-specific background mechanism. | Review the antibody's IHC-P dilution guidance, blocking and washes; adjust one workflow variable at a time while preserving the positive tissue control (general IHC practice). |
| Testis stains faintly while adrenal medulla stains strongly. | Different strengths can reflect the reported levels: medium in preleptotene spermatocytes and high in adrenal medullary cells (HPA tissue IHC). | Identify preleptotene spermatocytes before scoring. Compare nuclear signal with local background and the adrenal control; do not demand equal intensity across tissues (HPA tissue IHC; general IHC practice). |
| ICC-IF fluorescence looks nucleoplasmic, but IHC-P looks extranuclear. | The supported IF location is nucleoplasmic, while UniProt assigns PHOX2A to the nucleus (HPA subcellular ICC-IF; UniProt O14813). The assays and listed antibody validations differ (HPA antibodies). | Evaluate each assay with its own controls and antibody validation. For IHC-P, recheck cell identity, nuclear counterstain, and chromogenic background before accepting extranuclear signal (general IHC practice). |
Experiment evidence
IHC & IF images showing PHOX2A staining
A05780 · IHCImmunohistochemistry of PHOX2A in human brain tissue with PHOX2A antibody at 2.5 μg/mL.
A05780 · IFImmunofluorescence of PHOX2A in human brain tissue with PHOX2A antibody at 20 μg/mL.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000165462-PHOX2A/tissue/cerebral+cortex#imid_20535347
HPA · Adrenal glandHuman Protein Atlas IHC, Adrenal gland. Source: https://www.proteinatlas.org/ENSG00000165462-PHOX2A/tissue/adrenal+gland#imid_1161
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000165462-PHOX2A/tissue/colon#imid_20535257
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000165462-PHOX2A/tissue/liver#imid_20535274
HPA · ICC-IF SH-SY5YHuman Protein Atlas immunofluorescence, SH-SY5Y, antibody HPA053810; green = PHOX2A, blue = nucleus, red = microtubules.
HPA · ICC-IF SH-SY5YHuman Protein Atlas immunofluorescence, SH-SY5Y, antibody HPA053810; green = PHOX2A, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for PHOX2A IHC & IF
🧪Run adrenal gland first: medullary cells should show nuclear PHOX2A staining (HPA: High in medullary cells; UniProt O14813: nucleus). Use adipose tissue adipocytes as the negative comparison (HPA: Not detected in adipocytes); on the adrenal slide, compare medullary nuclei with neighboring cells that lack specific nuclear staining, without assuming every other cell type is negative (HPA: medullary cells scored High).
Positive control tissue: Adrenal gland (Medullary cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show PHOX2A in SH-SY5Y, U2OS, with annotated localisation: Nucleoplasm (supported) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control and an isotype control matched to the primary antibody’s host species and immunoglobulin class; use PHOX2A knockout tissue or cells, if available, as a biological negative. For chromogenic staining of the adrenal positive tissue, quench endogenous peroxidase and check background before scoring medullary nuclei (HPA: adrenal medullary cells High).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported in the supplied evidence, and the selected A05780 brain IHC caption does not state a fixative (caption: fixative unreported). Antigen retrieval dependency and conditions are also unreported; optimize retrieval on paraffin sections while assessing nuclear signal (UniProt O14813: nucleus; HPA subcellular: nucleoplasm). The supplied evidence does not establish whether frozen sections or IF/ICC are easier; endogenous peroxidase background in adrenal sections can complicate chromogenic interpretation, so assess it with the staining controls (HPA: adrenal medullary cells High).
HPA tissue IHC evidence for PHOX2A
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — High consistency between antibody staining and RNA expression data.). 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 | Medullary cells | High | Protein (IHC) | HPA → |
| Testis | Preleptotene spermatocytes | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Appendix | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Breast | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced PHOX2A IHC Tips
Troubleshoot PHOX2A staining in chromogenic paraffin section IHC using nuclear localisation, documented tissue patterns, and matched controls.
How should I retrieve PHOX2A in paraffin sections when nuclear staining is weak?
Start with Tris-EDTA at pH 9.0 for heat-induced retrieval at 95–98 °C for 20 min (page retrieval setting: nuclear antigen rule). Allow sections to cool in retrieval buffer before washing, and keep heating and cooling conditions consistent across comparison slides (standard IHC practice). PHOX2A is nuclear and has no annotated transmembrane segment, so assess whether signal resolves into nuclei rather than diffuse cytoplasm (UniProt O14813). If staining remains weak, compare a shorter retrieval time with the page setting on matched sections while holding antibody concentration and detection constant (standard IHC practice). Include an adrenal medulla section as a positive tissue control where available (HPA: high in medullary cells).
Could fixation explain absent PHOX2A staining in my paraffin sections?
PHOX2A-specific fixation sensitivity is unknown from the supplied evidence; the selected brain IHC caption does not state a fixative (A05780 tissue-IHC caption). Record fixative, fixation duration, block age, and processing history before comparing cases, because these variables can change antigen accessibility in paraffin IHC (standard IHC practice). Process a positive control alongside the sample and apply the same retrieval, antibody incubation, and detection conditions to both (standard IHC practice). The catalog image reports staining in human brain tissue at 2.5 µg/mL, but its caption cannot establish a fixation condition for your sections (A05780 tissue-IHC caption). If the control stains and the sample does not, review sample processing and tissue identity before changing retrieval (standard IHC practice).
What staining compartment should I accept as PHOX2A positive?
Score crisp nuclear staining as the expected PHOX2A pattern: UniProt assigns PHOX2A to the nucleus, and HPA reports supported nucleoplasmic localisation (UniProt O14813; HPA subcellular). Examine a counterstained section at high magnification to confirm that chromogen overlaps nuclei rather than adjacent cytoplasm or pigment (standard IHC practice). Strong diffuse cytoplasmic staining alone does not match the reported compartment and warrants review of antibody concentration, blocking, and detection controls (UniProt O14813; HPA subcellular). Check the positive control for the same nuclear pattern before calling an individual specimen negative (standard IHC practice). In adrenal gland, inspect medullary cells specifically because HPA reports high PHOX2A staining there (HPA tissue IHC).
How can I troubleshoot an apparent epitope mismatch without a mapped antibody epitope?
Do not assign this antibody to a particular PHOX2A epitope: none is supplied in the selected product caption (A05780 tissue-IHC caption). UniProt lists a 284-amino-acid chain, no annotated isoforms, no signal peptide, no propeptide, and no transmembrane segment (UniProt O14813). These annotations do not show whether fixation or retrieval preserves the antibody’s binding site, so compare staining across matched sections using the page retrieval setting (standard IHC practice; page retrieval setting). Use the catalog image concentration of 2.5 µg/mL only as a documented IHC reference, then titrate around it if your detection system or tissue processing differs (A05780 tissue-IHC caption; standard IHC practice). Judge each condition by nuclear signal in a positive control and background in a matched negative control (UniProt O14813; standard IHC practice).
How should I investigate PHOX2A by IF alongside this chromogenic IHC guide?
For IF/ICC, look for nucleoplasmic PHOX2A signal, consistent with UniProt nuclear localisation and HPA’s supported nucleoplasmic assignment (UniProt O14813; HPA subcellular). Pair PHOX2A with a separately validated marker of the expected cell type, and choose distinguishable fluorophores so cell identity and nuclear signal can be assessed together (standard IF practice). Examine an unstained section for autofluorescence, then place the weaker specific signal in a cleaner spectral channel and include single-label controls (standard IF practice). PHOX2A has no annotated transmembrane segment, so permeabilise sufficiently to let antibody reach the nuclear compartment while checking that nuclear morphology remains intact (UniProt O14813; standard IF practice). HPA lists ICC/IF images in SH-SY5Y and U2OS cells, but those images do not establish an IF fixation condition for your specimen (HPA subcellular).
What should I check when PHOX2A chromogen appears outside nuclei?
First inspect a no-primary control and the counterstain to separate detection-system deposits or endogenous pigment from true nuclear signal (standard IHC practice; UniProt O14813). For peroxidase-based chromogenic IHC, include a peroxidase block and assess whether deposits persist without primary antibody; these are general workflow checks (standard IHC practice). Reduce excessive antibody concentration or detection amplification if both the sample and negative control show widespread staining (standard IHC practice). The selected image used 2.5 µg/mL in human brain tissue, which is a reference point rather than proof that this concentration suits every processed section (A05780 tissue-IHC caption). Evaluate suspect signal against expected nucleoplasmic localisation and high medullary-cell staining in adrenal gland (HPA subcellular; HPA tissue IHC).
How should I quantify PHOX2A staining across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define positive staining as nuclear chromogen before scoring, consistent with PHOX2A’s nuclear and supported nucleoplasmic localisation (UniProt O14813; HPA subcellular). For a cell population, report the percentage of positive nuclei and an intensity-based H-score using the same scoring thresholds across slides (standard IHC practice). If the research question concerns spatial abundance, report positive nuclei per mm² of evaluable tissue instead (standard IHC practice). Normalise counts to the number of nuclei or evaluable tissue area in the specified compartment, and exclude folds, necrosis, and tissue edges by a prespecified rule (standard IHC practice). Keep adrenal medullary cells separate from other adrenal compartments when benchmarking against HPA’s high medullary-cell result (HPA tissue IHC).
How can I distinguish genuine PHOX2A staining from artefact?
Give greatest weight to discrete nuclear staining in the expected cell population, because PHOX2A is nuclear and HPA supports nucleoplasmic localisation (UniProt O14813; HPA subcellular). HPA reports high staining in adrenal medullary cells, medium staining in preleptotene spermatocytes, and no detected staining in several listed cell populations; interpret each named population separately (HPA tissue IHC). Treat staining confined to section edges or necrotic regions as suspect and compare it with an intact area on the same slide (standard IHC practice). If diffuse cytoplasmic deposits or no-primary staining are present, investigate endogenous pigment or enzyme activity and the chromogenic detection steps before assigning positivity (standard IHC practice). Confirm a questionable result with a matched positive control and consistent nuclear scoring criteria (standard IHC practice; UniProt O14813).
Boster reagents
Best PHOX2A / Paired mesoderm homeobox protein 2A IHC Antibodies
A05780 has human brain IHC and IF images (catalog: image captions) and lists Human, Mouse and Rat reactivity (catalog: reactivity).
Real IHC data
Anti-PHOX2A Antibody
Cat # A05780
A05780 is listed for IHC-P and IF (catalog: applications). Its images show IHC and IF in human brain tissue (catalog: IHC and IF image captions).
Which to pick: For paraffin-section IHC, choose A05780: IHC-P is listed (catalog: applications), and its human brain IHC image uses 2.5 μg/mL (catalog: IHC image caption); the fixative is unreported (catalog: IHC image caption). For IF, A05780 has a human brain image at 20 μg/mL (catalog: IF image caption); ICC validation and clonality are unreported (catalog: applications; catalog: clone). For cross-species planning, A05780 lists Human, Mouse and Rat reactivity (catalog: reactivity), while its supplied IHC and IF images show human brain tissue (catalog: IHC and IF image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry O14813 (PHX2A_HUMAN, Paired mesoderm homeobox protein 2A).
- Human Protein Atlas. PHOX2A tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. PHOX2A subcellular location (ICC-IF): Mainly localized to the nucleoplasm..
- Human Protein Atlas. PHOX2A antibody validation summary (3 antibodies).
- Anatomical characterisation of somatostatin-expressing neurons belonging to the anterolateral system. Scientific reports 2025 — PMC11923155.
- Deep sequencing of Phox2a nuclei reveals five classes of anterolateral system neurons. Proceedings of the National Academy of Sciences of the United States of America 2024 — PMC11161781.
- Phox2a Defines a Developmental Origin of the Anterolateral System in Mice and Humans. Cell reports 2020 — PMC7713706.
- Characterisation of deep dorsal horn projection neurons in the spinal cord of the Phox2a::Cre mouse line. Molecular pain 2022 — PMC9445510.
- PubMed PMID:8661014 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for PHOX2A (Paired mesoderm homeobox protein 2A). Expected localisation Nuclear localisation is expected; tissue staining compartment is unconfirmed (UniProt). Antigen retrieval: Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen). Validated IHC antibody: A05780 at 2.5 μg/mL (the product's own IHC validation image is shown). Controls (HPA): positive Adrenal gland (Medullary cells); negative Adipose tissue. 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 A05780); 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-PHOX2A, 2.5 μg/mL (datasheet A05780); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific); Caveat: A section lacking adrenal medullary cells may miss the positive population (HPA tissue IHC); Isoform / epitope: No annotated isoforms or processing; epitope effects unknown (UniProt); Regulation: Adrenal gland–enriched expression (HPA tissue RNA). Evidence: UniProt O14813, HPA tissue IHC · Enhanced, HPA ICC-IF · Mainly localized to the nucleoplasm., Datasheet A05780, PMC7713706. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/PHOX2A/immunohistochemistry-ihc-guide.