This website uses cookies to ensure you get the best experience on our website.
- Table of Contents
HLX / H2.0-like homeobox protein · IHC design guide
Design Immunohistochemistry for HLX
Plan chromogenic HLX IHC in paraffin sections with the catalog antibody's 1:100–1:300 dilution range (datasheet A30494). Compare the expected nuclear location (UniProt) with observed nuclear and cytoplasmic tissue staining, which has uncertain reliability (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
HLX 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 expected (UniProt); nuclear and cytoplasmic seen (HPA tissue IHC) | |
| Staining pattern | General nuclear and cytoplasmic staining (HPA tissue IHC) | |
| Antigen retrieval | Tris-EDTA pH 8.0 HIER, heat-mediated (datasheet A30494) | |
| Positive control | Adrenal gland+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 paraffin-section fixation consistent (standard IHC practice; not target-specific); Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A30494) | |
| Caveat | Uncertain staining; low agreement with RNA (HPA tissue IHC) | |
| Regulation | High in activated lymphocytes (UniProt) | |
| Isoform / epitope | No isoforms or cleavage; one 1–488 chain (UniProt) |
Section 1
Recommended HLX IHC & IF Protocols
Compare the catalog antibody’s IHC-P protocol with published HLX staining protocols for odontogenic cysts and mouse prostate cancer xenografts (PMC11857689; PMC13509987).
| Sample | Paraffin-embedded Human brain tissue; fixative not specified (datasheet A30494) |
| Fixation | Image fixative and duration unreported (datasheet A30494); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: Tris-EDTA pH 8.0 (datasheet A30494); 20 min, 95–100 °C (standard) |
| 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-HLX, 1:100 - 1:300 (datasheet A30494) |
| 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 | HLX-positive staining in glandular cells of adrenal gland (HPA tissue IHC: High). HPA tissue profile: General cytoplasmic and nuclear expression. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated Tris-EDTA, pH 8.0, for the catalog antibody (datasheet A30494). The published protocols use citrate-based retrieval (PMC11857689; PMC13509987).
Section 2
What Is the Expected HLX Staining Pattern?
HLX is a nuclear transcription factor with no transmembrane segment (UniProt Q14774). Expect nuclear staining in expressing cells, including bone marrow hematopoietic cells (HPA: High) and activated lymphocytes or monocytes (UniProt: tissue specificity). HPA also reports cytoplasmic and nuclear tissue staining, but rates its tissue IHC profile Uncertain because staining and RNA data have low consistency and external data contradict the reported location (HPA: tissue IHC reliability).
| Distinct nuclear staining in a subset of bone marrow hematopoietic cells, with restrained background. | This fits the reported high staining in bone marrow hematopoietic cells (HPA: High) and the nuclear location of HLX (UniProt Q14774). Record the fraction and intensity of positive cells separately; a whole-section average could hide a cell-restricted pattern (general IHC interpretation). HPA rates its tissue IHC profile Uncertain, so morphology alone does not establish antibody specificity (HPA: tissue IHC reliability). |
| Predominantly cytoplasmic staining, with little or no identifiable nuclear signal. | Treat this as ambiguous rather than an established HLX-positive result: UniProt places HLX in the nucleus (UniProt Q14774), while HPA reports general cytoplasmic and nuclear tissue staining with Uncertain reliability (HPA: tissue IHC). HPA separately approves nucleoplasm and cytosol localization in ICC-IF (HPA: subcellular), so cytoplasmic signal alone cannot prove an artefact. Check compartment boundaries and controls before scoring (general IHC practice). |
| Strong staining in a cell population selected as a low-expression or undetected comparator. | For example, adipocytes are reported as Not detected in adipose tissue (HPA: tissue IHC). Strong signal there warrants a cross-reactivity or endogenous detection-activity check (general IHC practice). It is a warning, not proof of either cause: HPA's tissue profile has Uncertain reliability (HPA: tissue IHC), and a comparator's reported level is specific to the listed cell population. |
| Diffuse color across cells and extracellular areas, obscuring nuclei. | A broad haze is difficult to interpret as cell-specific HLX staining (general IHC interpretation). Assess background with a no-primary control and inspect blocking, washes and chromogen development (general chromogenic IHC practice). Do not infer HLX expression from overall section color when the compartment and positive cells cannot be resolved; nuclear location is the UniProt expectation (UniProt Q14774). |
| No signal in bone marrow hematopoietic cells included as a positive comparator. | HPA reports High staining in these cells (HPA: tissue IHC), so first check that the comparator contains recognizable hematopoietic cells and that the staining run worked (general IHC practice). A blank result may reflect assay failure or sample variation; it cannot establish that all HLX expression is absent. Interpret cautiously because HPA rates the tissue profile Uncertain (HPA: tissue IHC reliability). |
💡Expected HLX appearanceA persuasive positive result is discrete nuclear staining in identifiable bone marrow hematopoietic cells (UniProt Q14774: nucleus; HPA: High), with intensity scored per cell; widespread unresolved cytoplasmic color or staining of an undetected comparator is suspect (HPA: tissue IHC; general IHC interpretation).
| Cell population chosen for interpretation | Bone marrow hematopoietic cells are reported High, whereas adipocytes in adipose tissue are Not detected (HPA: tissue IHC). Compare the named cells, not entire tissues; activated lymphocytes and monocytes are described as high-level contexts by UniProt (UniProt Q14774: tissue specificity). These sources do not establish an intensity threshold for every cell in a section. |
| Compartment and evidence strength | UniProt places HLX in the nucleus (UniProt Q14774); HPA tissue IHC reports both cytoplasmic and nuclear expression but is Uncertain (HPA: tissue IHC). HPA005968 is Uncertain for IHC and Approved for ICC (HPA: antibody validation). Require compartment-resolved, controlled interpretation before treating chromogenic cytoplasmic signal as HLX. |
| Topology and processing | HLX has no transmembrane segment or signal peptide, and its annotated chain spans residues 1–488 (UniProt Q14774). These annotations support interpreting it as a cellular transcription factor rather than expecting a membrane outline or secreted deposit (UniProt Q14774: function and topology). They do not identify an antibody epitope or predict antigen retrieval performance. |
| IF/ICC: what location is reported? | HPA approves nucleoplasm and cytosol localization in ICC-IF and lists images from HEK293, U-251MG and U2OS (HPA: subcellular). That cell-imaging observation can inform compartment assessment, but it does not upgrade the Uncertain tissue IHC profile or supply an IF/ICC protocol (HPA: subcellular; HPA: tissue IHC reliability). |
| Situation | Likely cause | Next action |
|---|---|---|
| Bone marrow comparator is blank. | The run may have failed, or the examined area may lack the relevant hematopoietic cells (general IHC practice); HPA reports these cells High (HPA: tissue IHC). | Confirm the cell population on the section, review run controls, then optimize the routine IHC workflow if needed (general IHC practice). Do not assign an HLX-negative score from a failed comparator. |
| Signal is mainly cytoplasmic. | Tissue IHC localization is disputed: UniProt says nucleus, while HPA reports cytoplasmic and nuclear staining with Uncertain reliability (UniProt Q14774; HPA: tissue IHC). | Re-examine nuclear boundaries and controls; report the observed compartment explicitly (general IHC interpretation). Keep a cytoplasm-only result provisional, while acknowledging HPA's approved cytosol ICC-IF location (HPA: subcellular). |
| Adipocytes stain strongly in the negative comparator. | HPA reports adipocytes as Not detected (HPA: tissue IHC); cross-reactivity or endogenous detection activity is possible (general IHC practice). | Run a no-primary detection control and inspect whether signal follows cells or the detection chemistry (general IHC practice). Reassess specificity before counting these cells as HLX positive. |
| All compartments show diffuse chromogen. | Nonspecific background or excessive development may obscure cell boundaries (general chromogenic IHC practice). | Check no-primary background, blocking, washes and development time (general chromogenic IHC practice). Score only cells with resolvable staining; UniProt's nuclear location provides the primary compartment reference (UniProt Q14774). |
| Few cells stain despite a clearly stained bone marrow comparator. | HLX expression can vary by cell population: UniProt describes low levels in normal B and T cells and high levels in activated lymphocytes and monocytes (UniProt Q14774: tissue specificity). | Identify and score the relevant cell populations separately, retaining intensity and positive-cell fraction (general IHC interpretation). Do not apply the bone marrow High designation to every cell in the test section (HPA: tissue IHC). |
| A membrane rim or extracellular deposit dominates the slide. | That distribution conflicts with nuclear localization and the absence of a transmembrane segment or signal peptide (UniProt Q14774). | Check morphology and detection controls, then withhold a positive HLX call unless compartment-resolved cellular signal is present (general IHC practice; UniProt Q14774: nucleus). |
Experiment evidence
IHC & IF images showing HLX staining
A30494 · IHCImmunohistochemical analysis of paraffin-embedded Human brain. Antibody was diluted at 1:100(4° overnight). High-pressure and temperature Tr
A30494 · IFImmunofluorescence analysis of MCF7 cells, using HLX1 Antibody. The picture on the right is blocked with the synthesized peptide.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000136630-HLX/tissue/cerebral+cortex#imid_2069904
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000136630-HLX/tissue/colon#imid_2069830
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000136630-HLX/tissue/liver#imid_2069831
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000136630-HLX/tissue/kidney#imid_2069840
HPA · ICC-IF HEK293Human Protein Atlas immunofluorescence, HEK293, antibody HPA005968; green = HLX, blue = nucleus, red = microtubules.
HPA · ICC-IF HEK293Human Protein Atlas immunofluorescence, HEK293, antibody HPA005968; green = HLX, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for HLX IHC & IF
🧪Run bone marrow first and score staining in its hematopoietic cells (HPA: High in bone marrow hematopoietic cells). Use adipose tissue as the negative tissue (HPA: Not detected in adipocytes); on the bone marrow slide, cells without target staining provide an internal background reference, but their identity is not specified by the supplied HPA row.
Positive control tissue: Adrenal gland (Glandular cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show HLX in HEK293, U-251MG, U2OS, with annotated localisation: Nucleoplasm (approved), Cytosol (approved) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control and a host-species-matched nonimmune IgG control, matched to the primary antibody’s isotype if monoclonal. Use HLX knockout material or an immunogen-peptide block as a biological specificity control (selected-SKU caption: peptide-block negative control), and quench endogenous peroxidase in bone marrow before chromogenic detection (standard IHC practice).
⚠️Feasibility: A target-specific fixation window and fixation effect are unreported, and the fixative in the selected-SKU paraffin-section caption is unreported (selected-SKU caption: fixative not stated). The caption used high-pressure, heated Tris-EDTA at pH 8.0 for retrieval and a 1:100 primary dilution overnight at 4 °C; it does not establish that retrieval is required (selected-SKU caption). Whether frozen sections or IF are easier is unreported; for bone marrow, endogenous peroxidase can create chromogenic background (standard IHC practice).
HPA tissue IHC evidence for HLX
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Uncertain — Low consistency between antibody staining and RNA expression data. Subcellular location is contradicted by external 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 → |
| Bone marrow | Hematopoietic cells | High | Protein (IHC) | HPA → |
| Caudate | Neuronal cells | High | Protein (IHC) | HPA → |
| Cerebellum | Cells in molecular layer | High | Protein (IHC) | HPA → |
| Cerebral cortex | Neuronal cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Colon | Endothelial cells | Not detected | Protein (IHC) | HPA → |
| Duodenum | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Esophagus | Squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
| Small intestine | Glandular cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced HLX IHC Tips
Troubleshoot HLX staining in paraffin sections by checking retrieval, nuclear localisation, tissue context and controls before interpreting chromogenic signal.
How should I optimise retrieval when HLX staining is weak?
Start with heat-mediated Tris-EDTA at pH 8.0 for paraffin sections (datasheet A30494). The selected brain image used high-pressure, high-temperature retrieval in that buffer, followed by antibody at 1:100 overnight at 4°C (caption A30494). If staining remains weak, compare a shorter and a longer heating interval on adjacent sections while holding antibody dilution, detection and development time constant (standard IHC practice). Inspect tissue morphology alongside signal, because excessive heating can damage sections and make apparent gains in staining difficult to interpret (standard IHC practice). Include a positive reference section in every comparison, and treat new cytoplasmic staining cautiously because tissue-IHC localisation has uncertain reliability (HPA tissue IHC: Uncertain).
Could fixation explain loss of HLX staining?
Target-specific sensitivity of HLX staining to fixation is unknown from the supplied evidence; the selected paraffin-section caption does not state a fixative (caption A30494). Record the actual fixative, fixation duration and processing history for each block before comparing staining intensity (standard IHC practice). On adjacent sections, keep Tris-EDTA retrieval at pH 8.0 and antibody incubation at 1:100 overnight at 4°C while assessing whether processing differences track the signal (datasheet A30494; caption A30494). Preserve section adhesion and nuclear detail when adjusting retrieval, and interpret a weak result against a concurrently stained positive reference rather than treating it as absent HLX (standard IHC practice).
How should I evaluate nuclear versus cytoplasmic HLX staining?
Score nuclear staining separately because HLX is annotated as a nuclear transcription factor (UniProt Q14774). Also record cytoplasmic signal without automatically rejecting it: HPA reports nucleoplasm and cytosol in its subcellular dataset, while its tissue-IHC profile describes general nuclear and cytoplasmic expression (HPA subcellular; HPA tissue IHC). The tissue-IHC assessment is Uncertain because antibody staining agrees poorly with RNA data and external localisation evidence conflicts (HPA tissue IHC). Compare nuclear boundaries against the counterstain, examine whether cytoplasmic colour follows tissue structures or section edges, and score compartments separately across matched sections (standard IHC practice). Require an independent control before assigning biological meaning to predominantly cytoplasmic chromogen (standard IHC practice; HPA tissue IHC: Uncertain).
Can isoforms or processing account for inconsistent HLX staining?
The supplied HLX record lists 0 isoforms, no annotated modified residues or glycosylation sites, and a single chain spanning residues 1–488 (UniProt Q14774). It also lists no signal peptide, propeptide or transmembrane segment, so the record supplies no processing feature that predicts a separate membrane-facing epitope (UniProt Q14774). The antibody's epitope coordinates are not supplied; therefore, an apparent compartment change cannot be assigned to epitope masking at a specific residue (supplied antibody evidence; UniProt Q14774). Compare serial sections with the same retrieval and detection conditions, then use the caption's immunogen-peptide preabsorption control where available to assess specificity of the observed stain (caption A30494; standard IHC practice).
How can I check HLX localisation by multiplex immunofluorescence?
For a separate IF experiment, pair HLX with a validated marker of the expected cell population; activated lymphocytes or monocytes are plausible populations from the expression record (UniProt Q14774). Choose fluorophores after checking unstained tissue autofluorescence, favouring a channel with clear separation from that background, and include single-colour controls for bleed-through (standard IF practice). HLX has no transmembrane segment and is annotated in the nucleus, while HPA also reports cytosol; use a controlled permeabilisation step to give antibodies access to these intracellular compartments (UniProt Q14774; HPA subcellular; standard IF practice). Compare HLX with a nuclear counterstain and score nuclear and cytosolic fluorescence separately, since tissue-IHC localisation carries Uncertain reliability (standard IF practice; HPA tissue IHC).
What controls distinguish HLX staining from chromogenic background?
Run a section without primary antibody to reveal detection-system background, and inspect endogenous peroxidase activity before interpreting DAB deposits (standard chromogenic IHC practice). The selected paraffin brain image includes a negative control made by preabsorbing the antibody with immunogen peptide; reproduce that control when reagent availability permits (caption A30494). If diffuse colour persists, compare blocking, wash stringency and chromogen development on adjacent sections while retaining Tris-EDTA retrieval at pH 8.0 (datasheet A30494; standard IHC practice). Assess pigment, folded tissue, section edges and necrotic regions under the counterstain before calling cells positive (standard IHC practice). Nuclear localisation supports a candidate HLX signal, but does not alone establish specificity (UniProt Q14774; standard IHC practice).
How should I quantify HLX across tissue sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and scoring region before reading slides, then report the percentage of positive nuclei and an H-score using fixed intensity categories (standard IHC practice). For dispersed positive cells, density per mm² is another useful measure if the sampled tissue area is recorded consistently (standard IHC practice). Normalise counts to eligible, intact cells or measured viable tissue area, and exclude folds, section edges and necrosis using the same rules for every specimen (standard IHC practice). Keep nuclear and cytoplasmic scores separate because UniProt places HLX in the nucleus, whereas HPA reports additional cytosolic staining and rates tissue-IHC reliability Uncertain (UniProt Q14774; HPA subcellular; HPA tissue IHC).
When is a positive HLX stain convincing?
A convincing candidate signal follows identifiable cells and includes nuclear staining, consistent with HLX's annotated transcription-factor localisation (UniProt Q14774; standard IHC practice). Evaluate the cell population against context: HLX is reported at low levels in normal B and T cells and at higher levels in activated lymphocytes and monocytes (UniProt Q14774). Treat staining confined to section edges, necrotic tissue, pigment or sites of endogenous enzyme activity as suspect until controls resolve it (standard IHC practice). The selected image's immunogen-peptide preabsorption control can test antibody-dependent colour, while a no-primary control checks detection background (caption A30494; standard IHC practice). Interpret cytoplasmic-only staining cautiously because tissue-IHC evidence has Uncertain reliability despite a cytosolic subcellular report (HPA tissue IHC; HPA subcellular).
Boster reagents
Best HLX / H2.0-like homeobox protein IHC Antibodies
The catalog lists anti-HLX antibodies for IHC and IF with human, mouse, and rat reactivity (catalog applications/reactivity); A30494 has pictured paraffin-section IHC and cell IF results (A30494 image captions).
Real IHC data
Anti-HLX/Hlx11 Antibody
Cat # A30494
A06177-1 lists IHC and IF applications and human, mouse, and rat reactivity, but provides no IHC or IF image caption (A06177-1 applications/reactivity/image alts). A30494 lists IHC, IF, and ICC and the same reactivity; its captions show paraffin-embedded human brain IHC and MCF7 cell IF (A30494 applications/reactivity/image captions).
Which to pick: For tissue IHC, pick A30494: its own caption documents paraffin-embedded human brain, 1:100 overnight at 4°C, and Tris-EDTA pH 8 retrieval; the fixative is unreported (A30494 IHC image caption). For IF/ICC, pick A30494 because its IF caption shows MCF7 cells and its application list includes both IF and ICC; it is described as polyclonal (A30494 IF image caption; A30494 applications; A30494 dilution_raw). For human, mouse, or rat samples, both SKUs list reactivity, while pictured results are available only for A30494 and clonality is unreported for A06177-1 (catalog reactivity/image alts; A30494 dilution_raw; A06177-1 clone).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q14774 (HLX_HUMAN, H2.0-like homeobox protein).
- Human Protein Atlas. HLX tissue IHC expression (reliability: Uncertain).
- Human Protein Atlas. HLX subcellular location (ICC-IF): Localized to the nucleoplasm and cytosol..
- Human Protein Atlas. HLX antibody validation summary (1 antibodies).
- Expression of Dlx-5 and HLX Proteins in Odontogenic Cysts. Life (Basel, Switzerland) 2025 — PMC11857689.
- A TBX2-HLX Regulatory Axis Is Associated with Advanced Prostate Cancer. Biomedicines 2026 — PMC13509987.
- The Hlx homeobox transcription factor is required early in enteric nervous system development. BMC developmental biology 2006 — PMC1564389.
- PubMed PMID:1676597 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
- PubMed PMID:16710414 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for HLX (H2.0-like homeobox protein). Expected localisation Nuclear expected (UniProt); nuclear and cytoplasmic seen (HPA tissue IHC). Antigen retrieval: Tris-EDTA pH 8.0 HIER, heat-mediated (datasheet A30494). Validated IHC antibody: A30494 at 1:100 - 1:300 (the product's own IHC validation image is shown). Controls (HPA): positive Adrenal gland (Glandular cells); negative Adipose tissue. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Paraffin-embedded Human brain tissue; fixative not specified (datasheet A30494); 2. Fixation: Image fixative and duration unreported (datasheet A30494); 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: Tris-EDTA pH 8.0 (datasheet A30494); 20 min, 95–100 °C (standard); 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-HLX, 1:100 - 1:300 (datasheet A30494); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep paraffin-section fixation consistent (standard IHC practice; not target-specific); Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A30494); Caveat: Uncertain staining; low agreement with RNA (HPA tissue IHC); Isoform / epitope: No isoforms or cleavage; one 1–488 chain (UniProt); Regulation: High in activated lymphocytes (UniProt). Evidence: UniProt Q14774, HPA tissue IHC · Uncertain, HPA ICC-IF · nucleoplasm and cytosol., Datasheet A30494, PMC11857689, PMC13509987. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/HLX/immunohistochemistry-ihc-guide.