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HEXA / Beta-hexosaminidase subunit alpha · IHC design guide
Design Immunohistochemistry for HEXA
Plan HEXA chromogenic IHC in paraffin sections using the catalog antibody at 0.5–1 μg/ml (datasheet: PA1787). Compare lysosomal staining in lung macrophages and gallbladder glandular cells, and interpret intensity with the reported low consistency between antibody staining and RNA expression (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
HEXA 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 | Lysosomal staining in several tissues (HPA tissue IHC) | |
| Staining pattern | Lysosomal signal in lung macrophages and gallbladder glandular cells (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen) | |
| Positive control | Gallbladder+4 more · see all | |
| Negative control | Bone marrow+4 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation conditions consistent across sections. (standard IHC practice; not target-specific) | |
| Caveat | Staining has low consistency with RNA expression (HPA tissue IHC) | |
| Regulation | Regulation unreported (UniProt) | |
| Isoform / epitope | 2 isoforms; the mature chain starts at residue 89 (UniProt) |
Section 1
Recommended HEXA IHC & IF Protocols
Compare the catalog antibody’s IHC-P protocol (datasheet) with one published HEXA paraffin-section method (PMC8371334).
| Sample | Tissue sections; selected-image fixative not specified (standard IHC workflow) |
| Fixation | Image fixative and duration unreported (datasheet PA1787); 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 citrate buffer, pH 6.0, 20 min at 95–98 °C (standard rule: cytoplasmic / membrane 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-HEXA, 0.5-1μg/ml (datasheet PA1787) |
| 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 | HEXA-positive staining in glandular cells of gallbladder (HPA tissue IHC: High). HPA tissue profile: Lysosomal expression in several tissues, most abundant in lung and gallbladder. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6.0 at 95–98 °C for 20 min (page retrieval rule); the published method uses two microwave cycles (PMC8371334).
Section 2
What Is the Expected HEXA Staining Pattern?
HEXA is a lysosomal protein without a transmembrane segment (UniProt P06865: location and topology). In paraffin section IHC, expect granular cytoplasmic staining, particularly in lung macrophages, gallbladder and prostate glandular cells, kidney tubular cells, and testis Leydig cells (HPA tissue IHC: High). HPA rates the tissue pattern Supported but reports low consistency between antibody staining and RNA expression, so interpret staining alongside controls (HPA tissue IHC: reliability).
| Discrete cytoplasmic granules in lung macrophages, with little nuclear or surface staining (HPA tissue IHC: High in lung macrophages; UniProt P06865: lysosome). | This fits lysosomal HEXA distribution (UniProt P06865: lysosome). Confirm the cellular pattern against a negative control and a second positive tissue, because HPA reports low antibody staining versus RNA consistency (HPA tissue IHC: reliability; general IHC practice). |
| Predominantly nuclear, sharply membranous, or extracellular staining in expected positive cells (UniProt P06865: lysosome; no transmembrane segment). | Those compartments do not fit the annotated location or topology (UniProt P06865: location and topology). Treat the pattern as suspect; inspect the negative control, counterstain, and tissue morphology before scoring it as HEXA (general IHC practice). |
| Strong signal chiefly in esophageal squamous cells or lymph node germinal center cells (HPA tissue IHC: Not detected in those cells). | This disagrees with the supplied HPA observations and may reflect antibody cross-reactivity or endogenous chromogen activity (HPA tissue IHC: Not detected; general IHC practice). A single discordant field does not prove either cause (general IHC practice). |
| Broad, diffuse color over stroma, lumina, or most cell types, without discernible granules (UniProt P06865: lysosome). | A uniform deposit is poorly aligned with a lysosomal target (UniProt P06865: lysosome). Compare a no-primary control and inspect blocking, washes, detection reagents, and development time as general IHC background checks (general IHC practice). |
| No detectable signal in lung macrophages or gallbladder glandular cells (HPA tissue IHC: High in both). | First suspect an assay or specimen problem, then assess whether the chosen antibody performs in IHC-P (general IHC practice). HPA's Supported reliability and low staining versus RNA consistency make an individual negative slide inconclusive (HPA tissue IHC: reliability). |
💡Expected HEXA appearanceCall a result consistent with HEXA when expected positive cells show granular cytoplasmic staining, strongest in HPA High populations such as lung macrophages or gallbladder glandular cells (UniProt P06865: lysosome; HPA tissue IHC: High); dominant nuclear, membranous, or diffuse tissue-wide color is suspect (UniProt P06865: location and topology; general IHC practice).
| Tissue and cell selection (HPA tissue IHC). | Use a High population such as lung macrophages, kidney tubular cells, or gallbladder glandular cells for a positive comparison (HPA tissue IHC: High). HPA reports low tissue RNA specificity, so a listed Not detected population is an observed IHC comparator, not proof that all its cells lack HEXA (HPA tissue IHC: RNA specificity and Not detected). |
| Antibody validation for paraffin section IHC (HPA antibodies). | HPA054583 is listed as IHC Supported; HPA018082 has no listed IHC validation in this payload (HPA antibodies: IHC status). Neither entry supplies a dilution, retrieval condition, or assurance that every specimen reproduces the tissue profile (HPA antibodies: supplied fields). |
| Processing and possible epitope position (UniProt P06865). | The annotated precursor has a signal peptide at residues 1–22, propeptide at 23–88, and chain at 89–529 (UniProt P06865: processing). Epitope location for the supplied antibodies is unspecified, so the effect of processing on either antibody's IHC signal cannot be predicted (HPA antibodies: supplied fields). |
| Isoforms and glycosylation (UniProt P06865). | Two isoforms and glycosylation sites at residues 115, 157, and 295 are annotated (UniProt P06865: isoforms and glycosylation). The payload does not map antibody epitopes to these features; do not assign a staining difference to a particular isoform or site (HPA antibodies: supplied fields). |
| IF/ICC Q&A: what localization is reported? (HPA subcellular ICC-IF). | Mainly vesicles, Supported; additional cytosol is Uncertain (HPA subcellular ICC-IF). This corroborates a punctate intracellular interpretation but comes from ICC-IF rather than paraffin section IHC (HPA subcellular ICC-IF; HPA tissue IHC). |
| Situation | Likely cause | Next action |
|---|---|---|
| Expected positive tissue has no stain (HPA tissue IHC: High in lung macrophages and gallbladder glandular cells). | Target-specific fixation effects are not established by the supplied assay evidence. Verify with a matched IHC source before attributing a result to fixation. | Run a known positive section with the same detection workflow; verify antibody identity and IHC validation, reagent activity, and section quality before calling the specimen negative (HPA antibodies: IHC status; general IHC practice). |
| All cells show weak, diffuse chromogen (UniProt P06865: lysosome). | Background from inadequate blocking, washes, or excess detection signal is possible (general IHC practice). A diffuse field does not match the expected lysosomal distribution (UniProt P06865: lysosome). | Inspect a no-primary control; adjust general IHC blocking, washing, antibody concentration, or chromogen development one variable at a time (general IHC practice). |
| Stain concentrates in nuclei or along cell borders (UniProt P06865: lysosome; no transmembrane segment). | Misidentified cells, counterstain confusion, or off-target staining may explain the discordant compartment (general IHC practice). | Recheck morphology and controls; compare with a second IHC-validated antibody if available, and avoid scoring the discordant compartment as HEXA without corroboration (HPA antibodies: IHC status; general IHC practice). |
| Strong signal appears in an HPA Not detected population, such as esophageal squamous cells (HPA tissue IHC: Not detected). | Cross-reactivity or endogenous detection activity is possible; HPA's observed negative is not an absolute biological absence claim (HPA tissue IHC: Not detected and reliability; general IHC practice). | Check no-primary and detection controls, including endogenous enzyme blocking when relevant to the chromogen, then compare a High tissue stained in the same run (HPA tissue IHC: High; general IHC practice). |
| Granules are present, but intensity varies among tissues (HPA tissue IHC: High, Medium, Low, and Not detected categories). | Cell populations and observed staining levels vary across the HPA tissue panel (HPA tissue IHC). HPA also reports low consistency with RNA expression (HPA tissue IHC: reliability). | Score the named cell population and subcellular pattern separately from intensity; compare equivalent regions and controls before interpreting variation as a biological change (HPA tissue IHC: cell-level observations; general IHC practice). |
| A retrieval change alters staining strength (general IHC practice). | Retrieval conditions can affect routine paraffin section IHC, but HEXA-specific retrieval sensitivity is unreported here (general IHC practice; HPA tissue IHC; UniProt P06865). | Document the condition and compare matched positive and negative controls under each setting; do not attribute the difference to a particular HEXA epitope without supporting data (general IHC practice; HPA antibodies: epitope unspecified). |
Experiment evidence
IHC & IF images showing HEXA staining
PA1787 · IHCAnti-HEXA antibody, PA1787, IHC(P) IHC(P):Human Intestinal Cancer Tissue
A00692-1 · IFIF analysis of HEXA using anti-HEXA antibody (A00692-1). HEXA was detected in an immunocytochemical section of U20S cells. Enzyme antigen re
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000213614-HEXA/tissue/cerebral+cortex#imid_15666063
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000213614-HEXA/tissue/colon#imid_15665997
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000213614-HEXA/tissue/liver#imid_15666006
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000213614-HEXA/tissue/kidney#imid_15665999
HPA · ICC-IF SK-MEL-30Human Protein Atlas immunofluorescence, SK-MEL-30, antibody HPA018082; green = HEXA, blue = nucleus, red = microtubules.
HPA · ICC-IF SK-MEL-30Human Protein Atlas immunofluorescence, SK-MEL-30, antibody HPA018082; green = HEXA, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for HEXA IHC & IF
🧪Run lung first and score macrophages for HEXA staining (HPA: High in lung macrophages). Use esophageal squamous epithelium as the negative tissue (HPA: Not detected); on the lung slide, cells without specific staining should show only background, although the HPA row does not establish that every nonmacrophage cell is negative.
Positive control tissue: Gallbladder (Glandular cells, HPA High)
Negative control tissue: Bone marrow (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show HEXA in SK-MEL-30, SiHa, U2OS, KOLF2.1J, with annotated localisation: Vesicles (supported) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control and an irrelevant IgG control matched to the primary antibody’s host species and, where applicable, isotype and clonality (standard IHC practice). Use HEXA-knockout material or a validated immunizing-peptide competition control as a biological specificity check; assess endogenous peroxidase in lung macrophages for chromogenic IHC and autofluorescence if using IF (standard IHC/IF practice).
⚠️Feasibility: A target-specific fixation window and the effect of antigen retrieval are unreported in the supplied evidence; optimize retrieval with the catalog antibody’s IHC-P protocol (datasheet: IHC-P protocol). The PA1787 paraffin-section caption identifies human intestinal cancer tissue but does not report a fixative (PA1787 tissue-IHC caption: fixative not stated). The evidence does not establish whether frozen sections or IF are easier; lung macrophage peroxidase activity and autofluorescence can complicate interpretation (standard IHC/IF practice).
HPA tissue IHC evidence for HEXA
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Supported — 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 |
|---|---|---|---|---|
| Gallbladder | Glandular cells | High | Protein (IHC) | HPA → |
| Kidney | Cells in tubules | High | Protein (IHC) | HPA → |
| Lung | Macrophages | High | Protein (IHC) | HPA → |
| Prostate | Glandular cells | High | Protein (IHC) | HPA → |
| Testis | Leydig cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Bone marrow | Hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Cerebellum | Cells in granular layer | Not detected | Protein (IHC) | HPA → |
| Cervix | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Esophagus | Squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
| Hippocampus | Glial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced HEXA IHC Tips
Troubleshoot HEXA staining in paraffin sections by checking retrieval, cellular pattern, controls and scoring before interpreting chromogenic signal.
How should I adjust retrieval when HEXA staining is weak or diffuse?
Start with citrate buffer at pH 6.0, heated to 95–98 °C for 20 min (page retrieval rule). If punctate staining is weak, compare a modestly longer incubation on matched sections while keeping antibody concentration and detection conditions constant; excessive retrieval can damage morphology or increase background (standard IHC practice). Include a gallbladder section with glandular cells or a lung section with macrophages as a positive reference, since both show high staining in the tissue atlas (HPA: high in gallbladder glandular cells and lung macrophages). Record retrieval conditions alongside staining intensity so a brighter but diffuse result is not mistaken for improved HEXA detection (UniProt P06865: lysosome; standard IHC practice).
Can fixation explain loss of HEXA staining in paraffin sections?
Target-specific fixation sensitivity for HEXA is unknown from the supplied evidence, so do not assign weak staining to a particular fixative or fixation duration (supplied HEXA evidence: no fixation comparison). The selected PA1787 tissue image identifies paraffin IHC in human intestinal cancer tissue but does not state its fixative (PA1787 IHC(P) caption). When troubleshooting, compare sections with documented, consistent processing and use the same retrieval and detection settings before changing the primary antibody concentration (standard IHC practice). Check whether morphology and a separately validated tissue control also deteriorate; widespread loss favors a processing problem over a HEXA-specific conclusion (standard IHC practice).
What cellular pattern should count as plausible HEXA staining?
Prioritize granular cytoplasmic staining compatible with lysosomes when assessing chromogenic HEXA signal (UniProt P06865: lysosome; HPA subcellular: vesicles supported). HPA also reports cytosolic localization as uncertain, so diffuse cytoplasmic color alone deserves more scrutiny than discrete vesicular staining (HPA subcellular: cytosol uncertain). Examine staining within the relevant cell population: lung macrophages and gallbladder glandular cells are high-staining references, whereas cerebellar granular-layer cells are reported as not detected (HPA tissue IHC). Score the cellular pattern at higher magnification after confirming that pigment, tissue folds and edge staining do not reproduce the apparent granules (standard IHC practice).
Could processing or isoforms change which HEXA epitopes are detected?
HEXA is synthesized with a signal segment at residues 1–22 and a propeptide at 23–88; the annotated chain spans 89–529 (UniProt P06865 processing). Its three annotated glycosylation sites are residues 115, 157 and 295, and two isoforms are listed (UniProt P06865 glycosylation and isoforms). If staining differs across specimens, establish the antibody’s immunogen and whether its epitope is present in the relevant processed chain and isoform before attributing the difference to expression (standard antibody validation practice; UniProt P06865 processing). An antibody signal does not distinguish alpha subunits in isozyme A from those in isozyme S without separate evidence (UniProt P06865 subunit composition).
How can IF help resolve an ambiguous chromogenic HEXA pattern?
Use IF as a separate localization check, pairing HEXA with a validated marker for the expected cell type, such as a macrophage marker when examining lung macrophages (HPA tissue IHC: high in lung macrophages; standard IF practice). Choose spectrally separated fluorophores after inspecting unstained tissue for autofluorescence, which can mimic puncta (standard IF practice). Because HEXA is a lysosomal protein without an annotated transmembrane segment, test permeabilization conditions that admit antibody to intracellular vesicles, while verifying that the marker retains its expected pattern (UniProt P06865 topology and localization; standard IF practice). Compare IF puncta with the chromogenic pattern on comparable material, without assuming IF conditions validate PA1787 paraffin staining (PA1787 IHC(P) caption; standard assay validation practice).
How do I reduce diffuse or granular background without erasing HEXA signal?
Run a section without primary antibody to identify detection-system background, and inspect unstained tissue for endogenous pigment before judging brown granules (standard chromogenic IHC practice). Block endogenous peroxidase before peroxidase-based DAB detection and optimize blocking and washes using matched sections (standard chromogenic IHC practice). Titrate the primary antibody and development time systematically; an apparent increase in vesicular staining is useful only if background remains controlled (standard IHC practice; HPA subcellular: vesicles supported). Compare a high-staining reference, such as gallbladder glandular cells, with a reported undetected population, such as bone-marrow hematopoietic cells, while recognizing that atlas observations are contextual controls rather than absolute negatives (HPA tissue IHC).
How should I score HEXA across regions with different cell composition? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and region before scoring, then record the percentage of positive cells and an intensity-based H-score using the same thresholds across slides (standard IHC quantification practice). Normalize positive-cell counts or stained area to the number of evaluable target cells or the measured region area in mm², respectively (standard IHC quantification practice). Separate lung macrophages from other lung cells, because HEXA staining is reported as high in macrophages and mixed-cell averages can reflect composition (HPA tissue IHC: high in lung macrophages; standard quantification practice). Exclude folds, necrosis and section edges using preset criteria, and report retrieval and detection settings with the scores (standard IHC quantification practice).
When is a strong HEXA stain likely to be misleading?
Treat vesicular cytoplasmic staining in an expected cell population as more persuasive than isolated nuclear or surface staining, given HEXA’s lysosomal annotation and supported vesicular location (UniProt P06865: lysosome and no transmembrane segment; HPA subcellular: vesicles supported). Check whether positive cells match the tissue context, such as lung macrophages or gallbladder glandular cells, while allowing for the atlas’s stated low consistency with RNA data (HPA tissue IHC). Edge effects, necrotic deposits, endogenous pigment and residual peroxidase can imitate chromogenic positivity; review morphology and the no-primary control before calling a result positive (standard IHC practice). Staining establishes antigen detection, not hexosaminidase activity or which alpha-containing isozyme is present (UniProt P06865 subunit composition; standard IHC interpretation).
Boster reagents
Best HEXA / Beta-hexosaminidase subunit alpha IHC Antibodies
Anti-HEXA antibodies have IHC data from human intestinal cancer tissue and IF data from U20S cells (catalog image captions); both list Human, Mouse and Rat reactivity (catalog: reactivity).
Real IHC data
Anti-HEXA Antibody ®
Cat # PA1787
Real IF data
Anti-HEXA Antibody ®
Cat # A00692-1
PA1787 is listed for IHC and has a paraffin-section image of human intestinal cancer tissue (catalog: applications; PA1787 image caption). A00692-1 is listed for IF/ICC and has an IF image of U20S cells (catalog: applications; A00692-1 image caption).
Which to pick: Choose PA1787 for paraffin-section IHC (catalog: applications; PA1787 image caption); the caption does not report the fixative (PA1787 image caption). Choose A00692-1 for IF/ICC (catalog: applications; A00692-1 image caption). For cross-species work, both list Human, Mouse and Rat reactivity (catalog: reactivity), while the supplied images show human samples only (catalog image captions); clonality is unreported (catalog: clone).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P06865 (HEXA_HUMAN, Beta-hexosaminidase subunit alpha).
- Human Protein Atlas. HEXA tissue IHC expression (reliability: Supported).
- Human Protein Atlas. HEXA subcellular location (ICC-IF): Mainly localized to vesicles. In addition localized to the cytosol..
- Human Protein Atlas. HEXA antibody validation summary (2 antibodies).
- Nucleus accumbens GLT-1a overexpression reduces glutamate efflux during reinstatement of cocaine-seeking but is not sufficient to attenuate reinstatement. Neuropharmacology 2018 — PMC6383073.
- Microglia-Specific Expression of HEXA and HEXB Leads to Poor Prognosis in Glioblastoma Patients. Frontiers in oncology 2021 — PMC8371334.
- Microsatellite instability in penile cancer: comparative analysis of primary tumors and metastases. Frontiers in urology 2026 — PMC13553218.
- Autophagic flux is impaired in the brain tissue of Tay-Sachs disease mouse model. PloS one 2023 — PMC10019743.
- PubMed PMID:2933746 — UniProt-cited evidence.
- PubMed PMID:2952641 — UniProt-cited evidence.
- PubMed PMID:1833974 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for HEXA (Beta-hexosaminidase subunit alpha). Expected localisation Lysosomal staining in several tissues (HPA tissue IHC). Antigen retrieval: Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen). Validated IHC antibody: PA1787 at 0.5-1μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Gallbladder (Glandular cells); negative Bone marrow. 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 PA1787); 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 citrate buffer, pH 6.0 , 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-HEXA, 0.5-1μg/ml (datasheet PA1787); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation conditions consistent across sections. (standard IHC practice; not target-specific); Caveat: Staining has low consistency with RNA expression (HPA tissue IHC); Isoform / epitope: 2 isoforms; the mature chain starts at residue 89 (UniProt); Regulation: Regulation unreported (UniProt). Evidence: UniProt P06865, HPA tissue IHC · Supported, HPA ICC-IF · Mainly localized to vesicles. In addition localized to the cytosol., Datasheet PA1787, PMC8371334. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/HEXA/immunohistochemistry-ihc-guide.