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HAX1 / HCLS1-associated protein X-1 · IHC design guide
Design Immunohistochemistry for HAX1
Plan HAX1 staining in paraffin sections using the IHC-validated antibody and its documented chromogenic protocol. Compare cytoplasmic staining in glandular and immune cells with matched controls, mindful that tissue staining has low consistency with RNA expression (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
HAX1 Immunohistochemistry Experimental Design Guide
Expected localisation, validated protocols, controls and antibodies — the at-a-glance facts below, then the full design guide.
Must know before staining
| Expected localisation | Cytoplasmic in most glandular and immune cells (HPA tissue IHC) | |
| Staining pattern | Glandular and immune cells show cytoplasmic staining (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A01495-2) | |
| Positive control | Appendix+4 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 | Antibody staining has low consistency with RNA expression (HPA tissue IHC) | |
| Regulation | Up-regulated in oral cancers (UniProt) | |
| Isoform / epitope | 6 isoforms; confirm antibody epitope coverage (UniProt) |
Section 1
Recommended HAX1 IHC & IF Protocols
The catalog antibody’s IHC-P protocol is paired with three published HAX1 IHC protocols covering NPC tumour tissues, a CRC tissue array, and paraffin-embedded tumour specimens (PMC9077140; PMC11217408; PMC8650040).
| Sample | Paraffin-embedded human esophageal squamous carcinoma tissue; fixative not specified (datasheet A01495-2) |
| Fixation | Image fixative and duration unreported (datasheet A01495-2); 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 A01495-2); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A01495-2) |
| Primary antibody | Rabbit anti-HAX1, 2-5 μg/ml (datasheet A01495-2) |
| Primary incubation | Overnight at 4 °C (datasheet A01495-2) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet A01495-2) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | HAX1-positive staining in glandular cells of appendix (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression in most glandular cells and in immune cells. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA pH 8.0 retrieval (datasheet: A01495-2); citrate at 95 °C for 30 min is a published alternative (PMC11217408: IHC methods).
Section 2
What Is the Expected HAX1 Staining Pattern?
In paraffin section IHC, expect HAX1 mainly in the cytoplasm of glandular and immune cells (HPA: tissue IHC profile; UniProt O00165: predominantly cytoplasmic). Several glandular cell populations show high staining, while some other sampled cell populations show none (HPA: tissue IHC). HAX1 has no transmembrane segment (UniProt O00165: topology). HPA rates the tissue IHC pattern Approved but reports low consistency with RNA expression (HPA: reliability).
| Glandular cells show cytoplasmic chromogenic staining, with strong signal in an appendix, duodenum, or stomach section. | This fits the reported compartment and cell type; glandular cells in those tissues are rated High (HPA: tissue IHC). Judge the cell distribution as well as intensity. An Approved IHC rating supports use of the pattern, but low agreement with RNA expression limits how confidently staining alone identifies HAX1 (HPA: reliability). |
| Staining appears only in nuclei, or forms a continuous extracellular deposit without discernible cytoplasmic signal. | Treat this as discordant with the usual IHC pattern and inspect controls and detection background (HPA: cytoplasmic tissue IHC profile; standard IHC practice). Nuclear HAX1 can occur when export is inhibited or after arsenite stress in vitro, so an isolated nuclear finding needs context before biological interpretation (UniProt O00165: subcellular location). |
| Adipocytes or oral mucosal squamous epithelial cells stain as strongly as glandular cells in a positive control. | Those specific cell populations are reported Not detected, whereas several glandular populations are High (HPA: tissue IHC). Check nonspecific antibody binding and endogenous detection activity before calling the staining HAX1 (standard IHC practice). A negative call applies to the listed cell population, not every cell in its tissue (HPA: tissue IHC). |
| Weak, even color covers cells, stroma, and spaces between cells, obscuring cell boundaries. | Diffuse color lacks the cell restricted cytoplasmic distribution described for HAX1 (HPA: tissue IHC profile). Compare a no primary control, blocking, washes, and detection chemistry to locate background (standard IHC practice). Do not score uniform haze as positive merely because HAX1 is described as ubiquitous (UniProt O00165: tissue specificity). |
| Glandular cells in a designated positive tissue show no signal while the counterstain and tissue morphology remain visible. | Appendix, duodenum, gallbladder, pancreas exocrine glands, rectum, small intestine, stomach, and thyroid gland contain listed High populations (HPA: tissue IHC). Confirm the relevant cells are present, then review the IHC validated antibody, retrieval, dilution, and detection controls (standard IHC practice); the payload gives no HAX1 specific retrieval condition. |
💡Expected HAX1 appearanceCall an IHC section positive when glandular cells show a discernible, potentially strong cytoplasmic reaction against a comparatively clear background (HPA: cytoplasmic profile; HPA: High in listed glandular populations); uniform haze or staining confined to cells listed Not detected is a suspect positive (HPA: tissue IHC; standard IHC practice).
| Tissue and cell choice (HPA: tissue IHC). | High staining is reported in appendix and duodenal glandular cells and pancreatic exocrine glandular cells; adipocytes and oral mucosal squamous epithelial cells are listed Not detected (HPA: tissue IHC). Compare the named cell populations, not whole tissues. |
| Compartment used for scoring (HPA: tissue IHC; UniProt O00165: subcellular location). | Predominantly cytoplasmic staining is the IHC expectation, despite additional organelle and membrane locations annotated by UniProt (HPA: tissue IHC profile; UniProt O00165: subcellular location). A fine organelle pattern need not be resolved by chromogenic IHC (standard IHC practice). |
| Antibody validation and interpretation limit (HPA: antibody validation; HPA: reliability). | HPA055141 is Approved for IHC, while the overall tissue pattern has low consistency with RNA expression (HPA: antibody validation; HPA: reliability). Use cell level morphology and controls when interpreting an unexpected signal (standard IHC practice). |
| Isoforms and antibody epitope (UniProt O00165: isoforms). | Six HAX1 isoforms are annotated (UniProt O00165: isoforms 1–6). Their recognition by the catalog antibody cannot be inferred because its epitope is absent from the supplied record; do not assign an unusual pattern to one isoform without separate evidence. |
| IF/ICC Q: What pattern is expected? (HPA: subcellular ICC-IF). | A: HPA reports a supported mitochondrial location in ICC-IF, with images from MCF-7, SK-MEL-30, and U2OS (HPA: subcellular ICC-IF). This is a cell imaging observation; score paraffin section IHC against its reported cytoplasmic tissue pattern (HPA: tissue IHC). |
| Situation | Likely cause | Next action |
|---|---|---|
| No staining in appendix or stomach glandular cells (HPA: High). | The positive cells may be absent from the examined area, or an IHC workflow step may have failed (HPA: tissue IHC; standard IHC practice). | Locate intact glands, check the positive control and detection reagents, then review retrieval and antibody dilution using the IHC validated antibody's instructions (standard IHC practice). |
| A section looks positive mainly in adipocytes (HPA: Not detected). | That cell level result conflicts with the HPA observation; nonspecific binding or detection background is possible (HPA: tissue IHC; standard IHC practice). | Inspect cell identity and a no primary control; assess blocking and washes before assigning HAX1 positivity (standard IHC practice). |
| Broad brown haze obscures cytoplasmic boundaries. | Background from antibody or chromogenic detection can prevent cell level scoring (standard IHC practice). | Review a no primary control, blocking, wash stringency, and detection time; rescore only where cells remain identifiable (standard IHC practice). |
| Only nuclei stain in an otherwise intact paraffin section. | This differs from the predominant cytoplasmic pattern, although conditional nuclear detection is annotated in vitro (HPA: tissue IHC; UniProt O00165: subcellular location). | Check a positive tissue control and nonspecific background; require experimental context before treating nuclear staining as a HAX1 localization change (standard IHC practice; UniProt O00165: subcellular location). |
| Glandular populations differ unexpectedly across tissues. | HPA reports both High and Not detected glandular populations, and its IHC pattern has low RNA consistency (HPA: tissue IHC; HPA: reliability). | Score each named cell population against its own HPA observation and review section quality and controls before interpreting the difference (HPA: tissue IHC; standard IHC practice). |
| A punctate ICC-IF image seems inconsistent with broad cytoplasmic IHC. | The two HPA observations use different imaging applications: mitochondrial ICC-IF and cytoplasmic tissue IHC (HPA: subcellular ICC-IF; HPA: tissue IHC). | Interpret each result with its application specific compartment and appropriate controls; avoid treating the ICC-IF image as a paraffin IHC scoring template (HPA: subcellular ICC-IF; HPA: tissue IHC). |
Experiment evidence
IHC & IF images showing HAX1 staining
A01495-2 · IHCIHC analysis of HAX1 using anti-HAX1 antibody (A01495-2). HAX1 was detected in a paraffin-embedded section of human esophageal squamous carc
A01495-2 · IHCIHC analysis of HAX1 using anti-HAX1 antibody (A01495-2). HAX1 was detected in a paraffin-embedded section of human colorectal adenocarcinom
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000143575-HAX1/tissue/cerebral+cortex#imid_15355922
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000143575-HAX1/tissue/colon#imid_15355864
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000143575-HAX1/tissue/liver#imid_15355857
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000143575-HAX1/tissue/kidney#imid_15355866
HPA · ICC-IF MCF-7Human Protein Atlas immunofluorescence, MCF-7, antibody HPA075289; green = HAX1, blue = nucleus, red = microtubules.
HPA · ICC-IF MCF-7Human Protein Atlas immunofluorescence, MCF-7, antibody HPA075289; green = HAX1, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for HAX1 IHC & IF
🧪Run duodenum first and score its glandular cells, which show High HAX1 staining (HPA: duodenum glandular cells, High); use fallopian tube glandular cells as the tissue negative (HPA: fallopian tube glandular cells, Not detected). On the positive slide, non-glandular cells can serve as a background comparison, but their HAX1 status is unreported in the supplied row, so do not treat them as confirmed internal negatives (HPA: duodenum glandular cells, High).
Positive control tissue: Appendix (Glandular cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show HAX1 in MCF-7, SK-MEL-30, U2OS, with annotated localisation: Mitochondria (supported) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control, a rabbit isotype control matched to the primary antibody’s immunoglobulin class, and HAX1-knockout material as a biological specificity control (catalog caption: rabbit anti-HAX1 antibody). Quench endogenous peroxidase and inspect inflammatory cells for background DAB signal when staining duodenum (standard IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected paraffin-section caption does not state a fixative (selected SKU A01495-2 tissue-IHC caption: fixative not stated). The caption uses heat-mediated retrieval in EDTA, pH 8.0, but does not establish that HAX1 staining depends on retrieval (selected SKU A01495-2 tissue-IHC caption). Whether frozen-section IHC or tissue IF is easier is unreported; duodenal inflammatory cells can contribute endogenous peroxidase background in chromogenic IHC (standard IHC practice).
HPA tissue IHC evidence for HAX1
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Low 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 |
|---|---|---|---|---|
| Appendix | Glandular cells | High | Protein (IHC) | HPA → |
| Duodenum | Glandular cells | High | Protein (IHC) | HPA → |
| Gallbladder | Glandular cells | High | Protein (IHC) | HPA → |
| Pancreas | Exocrine glandular cells | High | Protein (IHC) | HPA → |
| Rectum | Glandular cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Caudate | Glial cells | Not detected | Protein (IHC) | HPA → |
| Cerebellum | Cells in granular layer | Not detected | Protein (IHC) | HPA → |
| Cerebral cortex | Endothelial cells | Not detected | Protein (IHC) | HPA → |
| Fallopian tube | Glandular cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced HAX1 IHC Tips
These questions address HAX1 staining in paraffin sections with chromogenic IHC, with one question on IF.
Which retrieval conditions should I start with for HAX1 paraffin-section IHC?
Start with heat-mediated retrieval in EDTA at pH 8.0 for HAX1 paraffin-section IHC (datasheet A01495-2). The selected tissue image used that retrieval before incubation with 2 μg/ml primary antibody overnight at 4°C (datasheet A01495-2). If staining is weak, adjust heating duration on adjacent sections while keeping antibody concentration and detection conditions fixed (general IHC practice). Compare glandular and immune-cell staining with matched controls, since those compartments show cytoplasmic expression in tissue IHC (HPA: tissue IHC). Record retrieval duration and tissue integrity alongside staining intensity so a damaged section is not mistaken for improved antigen exposure (general IHC practice).
How should I troubleshoot weak HAX1 staining when tissue fixation varies?
HAX1-specific fixation sensitivity is unknown from the supplied evidence, and the selected paraffin-section caption does not state a fixative (datasheet A01495-2). Record the fixative and fixation duration for each specimen, then compare sections processed with the same EDTA retrieval at pH 8.0 and the same detection conditions (datasheet A01495-2; general IHC practice). Use a consistently stained control section in each run to separate a specimen-processing problem from a run-wide staining problem (general IHC practice). If weak signal tracks a fixation batch, repeat staining on matched material with controlled processing before assigning the difference to HAX1 abundance (general IHC practice).
Should HAX1 appear diffuse, punctate, or nuclear in chromogenic sections?
Expect predominantly cytoplasmic staining in chromogenic tissue sections, consistent with the tissue IHC profile and the protein record (HPA: tissue IHC; UniProt O00165). HAX1 also has annotated mitochondrial, endoplasmic-reticulum, vesicular, cortical, and nuclear locations, while mitochondrial localisation is supported by subcellular imaging (UniProt O00165; HPA: subcellular). Because DAB deposits can merge nearby structures in a thin section, do not call individual puncta mitochondrial without an independent compartment marker (general IHC practice). Evaluate nuclear staining separately: nuclear detection has been reported when export is inhibited or after arsenite stress in vitro (UniProt O00165). Compare any unusual pattern with intact neighboring cells and controls before interpreting a compartment shift (general IHC practice).
Could isoforms or epitope accessibility explain inconsistent HAX1 staining?
HAX1 has 6 annotated isoforms, but the supplied antibody caption does not map the recognized epitope or establish which isoforms it detects (UniProt O00165; datasheet A01495-2). The protein has no annotated transmembrane segment, and phosphorylation is annotated at Ser189 and Ser192; these facts alone do not establish an effect on IHC binding (UniProt O00165). When staining differs across specimens, first compare matched fixation, EDTA retrieval at pH 8.0, and detection conditions (datasheet A01495-2; general IHC practice). For an isoform-specific claim, obtain epitope mapping and independent isoform evidence before assigning a DAB pattern to one splice form (general IHC practice).
How can IF help resolve the HAX1 pattern seen by IHC?
Use IF as a complementary localisation check: tissue IHC reports cytoplasmic staining in glandular and immune cells, while subcellular imaging supports mitochondrial localisation (HPA: tissue IHC; HPA: subcellular). Multiplex HAX1 with a marker identifying the cell population being assessed and, when testing mitochondrial overlap, a mitochondrial marker (general IF practice). Choose fluorophores in channels with low tissue autofluorescence, and include unstained tissue to assess background before interpreting overlap (general IF practice). Because the antibody epitope is unmapped and HAX1 has intracellular locations, optimize permeabilisation for access to the relevant compartment rather than assuming a particular membrane side (datasheet A01495-2; UniProt O00165; general IF practice).
What should I check when HAX1 DAB staining looks widespread?
Check whether signal follows intact cell cytoplasm, as reported in tissue IHC, or instead coats section edges, damaged areas, and spaces outside cells (HPA: tissue IHC; general IHC practice). The selected paraffin-section procedure used 10% goat serum before 2 μg/ml primary antibody, followed by peroxidase detection with DAB (datasheet A01495-2). Include a no-primary control and an endogenous-peroxidase blocking step when evaluating diffuse DAB deposition (general IHC practice). Keep DAB development comparable across sections, and shorten development if background accumulates before cellular detail becomes clear (general IHC practice). Score only interpretable cells after checking the counterstain and tissue preservation (general IHC practice).
How should I quantify HAX1 staining across specimens? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and tissue region before scoring, because glandular and immune cells can both contribute cytoplasmic HAX1 staining (HPA: tissue IHC). For cellular staining, report the percentage of positive cells and an H-score from 0–300, using the same intensity thresholds for every section (general IHC practice). If counting positive cells by area, report density per mm² of viable tissue or of the defined compartment, and keep immune infiltrates separate from glandular cells (general IHC practice). Normalize comparisons to the same cell type, viable area, retrieval conditions, and image exposure or scanning settings (general IHC practice). Document the scoring rule and assess replicate sections before comparing groups (general IHC practice).
How do I distinguish genuine HAX1 staining from artefact?
A credible pattern places staining in intact cell cytoplasm, matching tissue IHC and the predominantly cytoplasmic protein annotation (HPA: tissue IHC; UniProt O00165). The selected antibody produced DAB staining in a paraffin section of human esophageal squamous carcinoma, but that image alone does not establish specificity in every tissue (datasheet A01495-2; general IHC practice). Treat staining confined to cut edges, necrotic areas, or no-primary controls as suspect, and check endogenous peroxidase before accepting diffuse DAB signal (general IHC practice). Investigate strong nuclear-only staining separately, since reported nuclear detection depends on particular experimental conditions in vitro (UniProt O00165). Interpret cell identity alongside staining intensity because immune cells may also stain (HPA: tissue IHC).
Boster reagents
Best HAX1 / HCLS1-associated protein X-1 IHC Antibodies
A01495-2 has real IHC data from human paraffin sections (A01495-2 image captions); the catalog provides no IF image or IF dilution (A01495-2 catalog).
Real IHC data
Anti-HAX1 Antibody ®
Cat # A01495-2
A01495-2 is listed for human IHC (A01495-2 applications and reactivity). Its captions document staining in paraffin sections of human esophageal squamous carcinoma, colorectal adenocarcinoma and lung cancer tissue (A01495-2 image captions).
Which to pick: Choose A01495-2 for human paraffin-section IHC; its rabbit antibody was used at 2 μg/ml after EDTA pH 8.0 heat retrieval in the documented images (A01495-2 image captions). No IF/ICC application or IF image is supplied for A01495-2, so there is no validated IF/ICC choice in this catalog (A01495-2 applications and image alts). No cross-species choice is documented because A01495-2 lists human reactivity only; its paraffin-section captions do not report the fixative (A01495-2 reactivity and image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry O00165 (HAX1_HUMAN, HCLS1-associated protein X-1).
- Human Protein Atlas. HAX1 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. HAX1 subcellular location (ICC-IF): Localized to the mitochondria..
- Human Protein Atlas. HAX1 antibody validation summary (2 antibodies).
- Cytoplasmic HAX1 Is an Independent Risk Factor for Breast Cancer Metastasis. Journal of oncology 2019 — PMC6481036.
- Extracellular vesicles rich in HAX1 promote angiogenesis by modulating ITGB6 translation. Journal of extracellular vesicles 2022 — PMC9077140.
- Energy stress promotes P-bodies formation via lysine-63-linked polyubiquitination of HAX1. The EMBO journal 2024 — PMC11217408.
- HAX1 maintains the glioma progression in hypoxia through promoting mitochondrial fission. Journal of cellular and molecular medicine 2021 — PMC8650040.
- PubMed PMID:9058808 — UniProt-cited evidence.
- PubMed PMID:18472110 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for HAX1 (HCLS1-associated protein X-1). Expected localisation Cytoplasmic in most glandular and immune cells (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A01495-2). Validated IHC antibody: A01495-2 at 2-5 μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Appendix (Glandular cells); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human esophageal squamous carcinoma tissue; fixative not specified (datasheet A01495-2); 2. Fixation: Image fixative and duration unreported (datasheet A01495-2); 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 A01495-2); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A01495-2); 8. Primary antibody: Rabbit anti-HAX1, 2-5 μg/ml (datasheet A01495-2); 9. Primary incubation: Overnight at 4 °C (datasheet A01495-2). Watch for: Fixation: Keep fixation consistent across sections. (standard IHC practice; not target-specific); Caveat: Antibody staining has low consistency with RNA expression (HPA tissue IHC); Isoform / epitope: 6 isoforms; confirm antibody epitope coverage (UniProt); Regulation: Up-regulated in oral cancers (UniProt). Evidence: UniProt O00165, HPA tissue IHC · Approved, HPA ICC-IF · mitochondria., Datasheet A01495-2, PMC9077140, PMC11217408. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/HAX1/immunohistochemistry-ihc-guide.