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HYI / Putative hydroxypyruvate isomerase · IHC design guide
Design Immunohistochemistry for HYI
Plan chromogenic IHC for HYI in paraffin sections using its reported cytoplasmic tissue pattern (HPA tissue IHC). This guide highlights staining controls, interpretation limits and the 4 annotated isoforms (HPA tissue IHC; UniProt).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
HYI 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 | Ubiquitous cytoplasmic tissue staining (HPA tissue IHC) | |
| Staining pattern | Cytoplasmic staining across many tissue cell types (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A14191-2) | |
| Positive control | Pancreas+4 more · see all | |
| Negative control | None in HPA (detected in all 44 tissues); use no-primary + isotype controls |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A14191-2) | |
| Caveat | Staining–RNA consistency is medium; verify specificity (HPA tissue IHC) | |
| Regulation | Low tissue specificity; no regulator established (HPA tissue IHC) | |
| Isoform / epitope | 4 isoforms; epitope effects are unknown (UniProt) |
Section 1
Recommended HYI IHC & IF Protocols
The catalog antibody has an IHC-P protocol (datasheet A14191-2); one published HYI IHC protocol provides a comparison (PMC11977543).
| Sample | Paraffin-embedded human melanomar tissue; fixative not specified (datasheet A14191-2) |
| Fixation | Image fixative and duration unreported (datasheet A14191-2); 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 A14191-2); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A14191-2) |
| Primary antibody | Rabbit anti-HYI, 2-5μg/ml (datasheet A14191-2) |
| Primary incubation | Overnight at 4 °C (datasheet A14191-2) |
| Detection | Streptavidin-biotin complex (SABC), DAB chromogen (datasheet A14191-2) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | HYI-positive staining in exocrine glandular cells of pancreas (HPA tissue IHC: High). HPA tissue profile: Ubiquitous cytoplasmic expression. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA retrieval at pH 8.0 (datasheet A14191-2). The published protocol reports antigen repair without specifying its conditions (PMC11977543).
Section 2
What Is the Expected HYI Staining Pattern?
HYI shows ubiquitous cytoplasmic tissue staining, with high signal in pancreatic exocrine glandular cells, placental trophoblastic cells, and seminal vesicle glandular cells (HPA tissue IHC). HPA rates the tissue pattern Approved, with medium agreement between staining and RNA data and external verification pending (HPA tissue IHC). UniProt annotates no transmembrane segment or subcellular location (UniProt Q5T013 topology); its topology does not independently establish the IHC compartment.
| Cytoplasmic stain in pancreatic exocrine glandular cells, placental trophoblastic cells, or seminal vesicle glandular cells. | This matches the reported high staining in those cell populations (HPA tissue IHC). Assess the named cells, because a tissue-wide impression can obscure whether the expected cells carry the signal (general IHC practice). |
| Predominantly nuclear stain, with little cytoplasmic signal in the expected cells. | A predominantly nuclear IHC pattern differs from HPA's ubiquitous cytoplasmic tissue profile (HPA tissue IHC). Treat it as an unexpected result and check specificity and chromogen distribution; HPA's ICC-IF locations do not establish nuclear IHC staining (HPA subcellular). |
| Strong stain in a population reported as low, while expected high-staining cells remain weak. | This reverses the reported contrast between, for example, pancreatic exocrine glandular cells and smooth muscle cells (HPA tissue IHC). Check cell identification, possible antibody cross-reactivity, and endogenous detection activity (general IHC practice). A low HPA level is not a negative control (HPA tissue IHC). |
| Similar chromogen haze across cells, extracellular areas, and the section edge. | A uniform haze is hard to reconcile with an interpretable cytoplasmic cell pattern (HPA tissue IHC). Review the no-primary control, detection background, washing, and antibody concentration before assigning cellular HYI positivity (general IHC practice). |
| No visible signal in a pancreatic, placental, or seminal vesicle section containing the named cells. | Those cell populations were reported as high staining (HPA tissue IHC), so an absent signal calls for assay checks. Confirm that the expected cells are present, then review retrieval, antibody and detection performance, and controls (general IHC practice). |
💡Expected HYI appearanceCall an IHC result concordant when the named high-staining glandular or trophoblastic cells show clear cytoplasmic signal (HPA tissue IHC); widespread nuclear signal or cell-independent haze is unexpected against that profile (HPA tissue IHC; general IHC practice).
| Tissue and cell selection | HPA reports high staining in pancreatic exocrine glandular cells, placental trophoblastic cells, and seminal vesicle glandular cells (HPA tissue IHC). Its low entries, including smooth muscle cells and fibroblasts, offer contrasts but are not proven HYI-negative populations (HPA tissue IHC). |
| Strength of tissue evidence | The tissue IHC reliability is Approved, with medium staining-to-RNA consistency and external verification pending (HPA tissue IHC). Use the reported pattern as a benchmark, while treating an unexpected result as a reason to verify staining and cell identity. |
| IHC versus ICC-IF compartment evidence | HPA describes tissue IHC as ubiquitously cytoplasmic (HPA tissue IHC). Its ICC-IF record places HYI mainly at the plasma membrane and in the cytosol, with additional Golgi localization (HPA subcellular). These observations come from different preparations. |
| Topology and annotated processing | UniProt annotates no transmembrane segment or signal peptide and lists one chain spanning residues 1–277 (UniProt Q5T013 topology and processing). Those annotations give no basis for assuming a membrane-spanning IHC pattern or a cleaved staining product. |
| Isoforms and epitope coverage | UniProt lists four HYI isoforms (UniProt Q5T013 alternative splicing). The supplied record does not locate the antibody epitope or establish recognition of each isoform, so differences between specimens cannot be assigned to an isoform from this evidence. |
| Situation | Likely cause | Next action |
|---|---|---|
| Expected high-staining cells are present, but the IHC slide is blank. | Possible assay failure or an unhelpful retrieval or detection setting (general IHC practice); high staining is reported for the named cells (HPA tissue IHC). | Check the positive-control section, antibody and detection steps, and the retrieval conditions used in the IHC workflow (general IHC practice). Avoid assigning HYI absence from a failed control. |
| The slide has a diffuse brown background. | Residual detection activity, concentrated antibody, or insufficient washing can raise background in chromogenic IHC (general IHC practice). | Compare the no-primary control, review endogenous activity blocking and washes, and optimize antibody concentration (general IHC practice). Score only a resolved cellular pattern against the reported cytoplasmic profile (HPA tissue IHC). |
| Nuclei dominate the signal. | This differs from the reported cytoplasmic tissue profile (HPA tissue IHC); background or an unintended staining reaction is possible (general IHC practice). | Check counterstain and no-primary controls, then confirm the compartment in another section or with an independent specificity control (general IHC practice). |
| Low-reported cells stain more strongly than the expected high-reported cells. | Misidentified cells, cross-reactivity, or endogenous detection activity may explain the reversal (general IHC practice); HPA reports different levels for these populations (HPA tissue IHC). | Recheck morphology and controls, and compare another reported high-staining tissue if available (general IHC practice; HPA tissue IHC). Do not treat an HPA low category as zero expression. |
| A specimen differs from the reported HPA pattern despite usable controls. | HPA reports medium staining-to-RNA consistency and pending external verification (HPA tissue IHC); the supplied record cannot identify the reason for this specimen's difference. | Document the cell type, compartment, and relative intensity, then seek independent confirmation before interpreting the difference as HYI biology (general IHC practice). |
| Q: What pattern should be checked in IF/ICC? | HPA reports approved plasma membrane and cytosol localization, plus additional Golgi localization in ICC-IF (HPA subcellular). | A: Assess those compartments in the separate IF/ICC guide (HPA subcellular). The tissue IHC result is reported as cytoplasmic (HPA tissue IHC). |
Experiment evidence
IHC & IF images showing HYI staining
A14191-2 · IHCIHC analysis of HYI using anti-HYI antibody (A14191-2). HYI was detected in paraffin-embedded section of human melanomar tissue. Heat mediat
A14191-2 · IHCIHC analysis of HYI using anti-HYI antibody (A14191-2). HYI was detected in paraffin-embedded section of human rectal cancer tissue. Heat me
A14191-2 · IFIF analysis of HYI using anti-HYI antibody (A14191-2). HYI was detected in immunocytochemical section of A431 cells. Enzyme antigen retrieva
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000178922-HYI/tissue/cerebral+cortex#imid_10340561
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000178922-HYI/tissue/colon#imid_10340495
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000178922-HYI/tissue/liver#imid_10340496
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000178922-HYI/tissue/kidney#imid_10340497
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA040092; green = HYI, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA040092; green = HYI, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for HYI IHC & IF
🧪Run pancreas first: exocrine glandular cells should stain (HPA: High in pancreatic exocrine glandular cells). HPA detects HYI in all 44 scored tissues, so there is no supported negative tissue; use no-primary and isotype controls, and treat any non-exocrine cells on the slide as internal negatives only if independently shown to lack HYI, with staining at background level (HPA: no negative tissue rows).
Positive control tissue: Pancreas (Exocrine glandular cells, HPA High)
Negative control tissue: None in HPA: HYI is detected in all 44 scored tissues. Use a no-primary (secondary-only) and an isotype control instead.
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show HYI in A-431, U-251MG, U2OS, with annotated localisation: Plasma membrane (approved), Cytosol (approved) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control; a rabbit IgG control matched to the primary antibody’s class and clonality where applicable (caption: rabbit anti-HYI); and HYI knockout material or a validated peptide-block control as a biological negative. For pancreatic DAB staining, block endogenous peroxidase and check for endogenous biotin when using the caption’s biotin-based detection (caption: biotinylated secondary and DAB).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected A14191-2 paraffin-section caption does not state a fixative (caption: fixative unreported). The demonstrated paraffin workflow uses heat retrieval in EDTA at pH 8.0; no matched evidence establishes that frozen sections or IF are easier (caption: EDTA retrieval; HPA: ICC-IF images in A-431, U-251MG and U2OS). Process pancreas promptly to limit exocrine autolysis, which can impair tissue interpretation (standard IHC practice).
HPA tissue IHC evidence for HYI
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Medium consistency between antibody staining and RNA expression data. Pending external verification.). Rows are taken directly from the HPA tissue chart — click any row's HPA link to view the source.
Positive expression · recommended positive controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Pancreas | Exocrine glandular cells | High | Protein (IHC) | HPA → |
| Placenta | Trophoblastic cells | High | Protein (IHC) | HPA → |
| Seminal vesicle | Glandular cells | High | Protein (IHC) | HPA → |
| Stomach | Glandular cells | Medium | Protein (IHC) | HPA → |
| Adipose tissue | Adipocytes | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| None in HPA: HYI is detected in all 44 scored tissues. Use a no-primary (secondary-only) and an isotype control instead. | ||||
Section 3
Advanced HYI IHC Tips
Troubleshoot HYI staining in paraffin sections using the catalog antibody’s tissue result and protein localisation data; assess IF separately.
Which retrieval conditions should I start with for HYI paraffin IHC?
Start with heat-mediated retrieval in EDTA at pH 8.0 for paraffin sections (datasheet A14191-2). The selected tissue image used this condition before 10% goat-serum blocking and overnight incubation at 4°C with 2 µg/ml primary antibody (datasheet A14191-2). If staining is weak, check that section heating and cooling were consistent across the run before changing retrieval conditions (standard IHC practice). A different buffer or pH is a fallback to test on matched sections, with morphology and background assessed alongside signal; the supplied tissue result does not establish that alternative for this antibody (datasheet A14191-2).
How should I troubleshoot weak HYI staining when fixation history is uncertain?
HYI-specific sensitivity to fixation is unknown: the selected paraffin-section caption does not state a fixative (datasheet A14191-2). Record the available fixation and processing history, then compare sections processed alike before attributing a weak result to fixation (standard IHC practice). Keep retrieval at EDTA pH 8.0 and the primary concentration at 2 µg/ml while assessing whether staining and tissue morphology change together (datasheet A14191-2). If processing history differs between samples, include a consistently processed reference section in each run and interpret intensity comparisons cautiously (standard IHC practice); HPA staining patterns do not establish a fixation effect for this antibody (HPA tissue IHC).
Where should HYI signal appear, and how should compartment differences be checked?
Expect predominantly cytoplasmic tissue staining as a reference pattern, while allowing membrane-associated signal where cell boundaries can be resolved (HPA tissue IHC; HPA subcellular). In cell imaging, HYI is approved at the plasma membrane and cytosol, with an additional Golgi location; UniProt provides no subcellular annotation and reports no transmembrane segment (HPA subcellular; UniProt Q5T013). For chromogenic sections, compare candidate membrane staining with an adjacent counterstained section and inspect cytoplasmic signal in intact cells at the same exposure or viewing conditions (standard IHC practice). Strong nuclear-only staining warrants scrutiny of controls and section quality before assigning it to HYI (HPA subcellular; standard IHC practice).
Can this IHC result distinguish HYI isoforms or identify the stained epitope?
Do not assign a paraffin-section signal to one splice form from this stain alone: HYI has 4 listed isoforms, and the supplied antibody caption does not map its epitope (UniProt Q5T013; datasheet A14191-2). UniProt lists a single 1–277 chain, no signal peptide or propeptide, and no annotated glycosylation or modified residues (UniProt Q5T013). Those features do not show which sequence the catalog antibody recognises in fixed sections (UniProt Q5T013; datasheet A14191-2). For isoform-specific conclusions, first establish the immunogen or mapped epitope and compare it with each isoform sequence; use orthogonal evidence to support any resulting assignment (standard IHC practice).
How should I adapt HYI localisation checks to multiplex IF?
Treat IF as a separate assay: the selected antibody evidence describes chromogenic staining in paraffin sections, while the listed cell images support HYI localisation without establishing this catalog antibody’s IF conditions (datasheet A14191-2; HPA subcellular). Pair HYI with a validated marker of the expected cell type, such as a trophoblastic-cell marker in placenta, and include single-stain controls for channel separation (HPA: High in trophoblastic cells; standard IF practice). Choose a fluorophore channel after checking tissue autofluorescence and detector bleed-through (standard IF practice). If the epitope faces the cytosol, assess permeabilisation; if it is exposed at the cell surface, compare staining without permeabilisation, because epitope orientation is not supplied (UniProt Q5T013 topology; standard IF practice).
What should I check when HYI DAB staining is diffuse or widespread?
Widespread cytoplasmic staining can be plausible because HPA describes ubiquitous HYI expression, so assess cell outlines and staining intensity before calling it background (HPA tissue IHC). Compare a no-primary control with the test section to identify secondary-reagent or chromogen signal, and inspect tissue edges and damaged areas separately (standard IHC practice). The selected image used a biotinylated goat anti-rabbit secondary, streptavidin-biotin detection and DAB; assess background from that detection chain in the same tissue (datasheet A14191-2; standard IHC practice). Include a peroxidase block for DAB detection as a general workflow control, and compare sections developed for the same time (standard IHC practice).
How can I quantify HYI IHC across samples with different cell composition? ⚠ ANSWER MARKED FOR VERIFICATION
Define the scored compartment and cell population before measuring HYI, using cytoplasmic staining as the tissue reference pattern (HPA tissue IHC; standard IHC practice). Report the percentage of positive cells and staining intensity, or calculate an H-score from the proportions at intensity grades 0–3, giving a 0–300 scale (standard IHC practice). Normalise counts to the number of evaluable cells in the chosen population, or report positive-cell density per mm² of viable tissue when cell counts are impractical (standard IHC practice). Keep retrieval, DAB development, counterstain and scoring thresholds consistent, and document excluded necrotic or damaged regions (standard IHC practice).
How do I distinguish convincing HYI staining from an artefact?
Look for reproducible cellular staining with a predominantly cytoplasmic pattern, then assess membrane-associated signal against intact cell boundaries (HPA tissue IHC; HPA subcellular). Strong staining in pancreatic exocrine glandular cells or placental trophoblastic cells can support a run, while low staining in oral-mucosal squamous epithelial cells offers a comparison; neither pattern validates an individual antibody result by itself (HPA tissue IHC; standard IHC practice). Treat nuclear-only signal, section-edge staining and necrotic regions as findings to investigate with matched controls (HPA subcellular; standard IHC practice). A no-primary control and an adequate peroxidase block help distinguish antibody-associated DAB from detection background or endogenous enzyme activity (standard IHC practice).
Boster reagents
Best HYI / Putative hydroxypyruvate isomerase IHC Antibodies
A14191-2 has paraffin-section IHC images from human melanoma and rectal cancer tissue and an IF image from A431 cells (IHC and IF image captions). Catalog reactivity lists Human, Mouse and Rat (catalog reactivity).
Real IHC data
Anti-HYI Antibody ®
Cat # A14191-2
A14191-2 is listed for IHC and IF/ICC (catalog applications). Its IHC images show human melanoma and rectal cancer paraffin sections; its IF image shows A431 cells (IHC and IF image captions).
Which to pick: Choose A14191-2 for tissue IHC using paraffin sections: its IHC captions report EDTA retrieval at pH 8.0 and 2 μg/ml antibody; the fixative is unreported (IHC image captions). Choose the same SKU for IF/ICC: its IF caption shows A431 cells stained at 5 μg/ml (IF image caption). For cross-species work, the catalog lists Human, Mouse and Rat reactivity, while the supplied IHC images show human tissue only; clonality is unreported (catalog reactivity; IHC image captions; catalog clone field).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q5T013 (HYI_HUMAN, Putative hydroxypyruvate isomerase).
- Human Protein Atlas. HYI tissue IHC expression (reliability: Approved).
- Human Protein Atlas. HYI subcellular location (ICC-IF): Mainly localized to the plasma membrane and cytosol. In addition localized to the Golgi apparatus..
- Human Protein Atlas. HYI antibody validation summary (1 antibodies).
- Metabolomics- and Proteomics-Based Disease Diagnostic Classifier Model for the Prediction and Diagnosis of Colorectal Carcinoma. Journal of proteome research 2025 — PMC11977543.
- PubMed PMID:16710414 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
- PubMed PMID:19722193 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for HYI (Putative hydroxypyruvate isomerase). Expected localisation Ubiquitous cytoplasmic tissue staining (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A14191-2). Validated IHC antibody: A14191-2 at 2-5μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Pancreas (Exocrine glandular cells); negative None in HPA (detected in all 44 tissues); use no-primary + isotype controls. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human melanomar tissue; fixative not specified (datasheet A14191-2); 2. Fixation: Image fixative and duration unreported (datasheet A14191-2); 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 A14191-2); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A14191-2); 8. Primary antibody: Rabbit anti-HYI, 2-5μg/ml (datasheet A14191-2); 9. Primary incubation: Overnight at 4 °C (datasheet A14191-2). Watch for: Fixation: Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A14191-2); Caveat: Staining–RNA consistency is medium; verify specificity (HPA tissue IHC); Isoform / epitope: 4 isoforms; epitope effects are unknown (UniProt); Regulation: Low tissue specificity; no regulator established (HPA tissue IHC). Evidence: UniProt Q5T013, HPA tissue IHC · Approved, HPA ICC-IF · Mainly localized to the plasma membrane and cytosol. In addition…, Datasheet A14191-2, PMC11977543. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/HYI/immunohistochemistry-ihc-guide.