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- Table of Contents
HK3 / Hexokinase-3 · IHC design guide
Design Immunohistochemistry for HK3
Plan HK3 staining in paraffin sections using the IHC-validated antibody (datasheet A05145-3). Bone marrow hematopoietic cells and lung macrophages show high cytoplasmic staining, providing cell-specific reference patterns for interpretation (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
HK3 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 staining in hematopoietic and immune cells (HPA tissue IHC) | |
| Staining pattern | Selective cytoplasmic staining in hematopoietic and immune cells (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A05145-3) | |
| Positive control | Bone marrow+3 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 | Immune-cell abundance can change apparent tissue signal (HPA tissue IHC) | |
| Regulation | Cell-type-dependent expression (HPA tissue IHC) | |
| Isoform / epitope | One 1–923 chain; no isoforms or processing annotated (UniProt) |
Section 1
Recommended HK3 IHC & IF Protocols
The catalog antibody’s IHC-P protocol is paired with 4 published HK3 IHC protocols covering human tissue and rat lung (PMC11956552; PMC9618622; PMC13616702; PMC11076449).
| Sample | Paraffin-embedded human liver tissue; fixative not specified (datasheet A05145-3) |
| Fixation | Image fixative and duration unreported (datasheet A05145-3); 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 A05145-3); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A05145-3) |
| Primary antibody | Rabbit anti-HK3, 2-5 μg/ml (datasheet A05145-3) |
| Primary incubation | Overnight at 4 °C (datasheet A05145-3) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet A05145-3) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | HK3-positive staining in hematopoietic cells of bone marrow (HPA tissue IHC: High). HPA tissue profile: Selective cytoplasmic expression in hematopoietic cells and subsets of immune cells. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA pH 8.0 retrieval for the catalog antibody (datasheet A05145-3); the rat lung study used citrate buffer with a different antibody (PMC13616702).
Section 2
What Is the Expected HK3 Staining Pattern?
HK3 should appear as cytoplasmic staining in hematopoietic cells and subsets of immune cells (HPA tissue IHC: selective cytoplasmic expression). Expect the strongest staining in bone marrow hematopoietic cells, lung macrophages and splenic red-pulp cells (HPA tissue IHC: High). This pattern has Enhanced IHC reliability, reflecting consistency with RNA expression (HPA tissue IHC: Enhanced). HK3 has no annotated transmembrane segment or signal peptide (UniProt P52790 topology and processing).
| Strong cytoplasmic staining in bone marrow hematopoietic cells, lung macrophages or splenic red-pulp cells. | This matches the reported HK3-positive cell types and compartment (HPA tissue IHC: High; selective cytoplasmic expression). Judge staining in the identified cells, since each tissue contains other cell types for which this record gives no equivalent positive call. |
| Predominantly nuclear or sharply membrane-bound staining in otherwise plausible positive cells. | Treat this as an unexpected pattern: HPA reports cytoplasmic HK3, and UniProt annotates no transmembrane segment (HPA tissue IHC: cytoplasmic; UniProt P52790 topology). Check controls and repeat the interpretation before scoring those cells as HK3-positive (general IHC practice). |
| Strong staining of bronchial respiratory epithelium, adipocytes or the listed glandular cells. | These cell populations are reported as Not detected, so staining there warrants checking antibody specificity or endogenous chromogen activity (HPA tissue IHC: bronchus, adipose tissue, adrenal gland and appendix). Do not extend that negative call to every cell in those tissues. |
| Color spread broadly across the section, including areas outside identifiable cells. | A diffuse deposit does not establish the selective cellular pattern reported for HK3 (HPA tissue IHC: selective cytoplasmic expression). Compare a no-primary control and review blocking, washing and detection conditions to assess nonspecific or endogenous signal (general IHC practice). |
| No staining in an adequately represented bone marrow hematopoietic-cell or lung macrophage control. | That conflicts with the reported High staining in those cells (HPA tissue IHC: bone marrow hematopoietic cells; lung macrophages). Review the control's cell content and the IHC staining workflow before calling an experimental section negative (general IHC practice). |
💡Expected HK3 appearanceCall the slide positive when identifiable hematopoietic cells or immune-cell subsets show selective cytoplasmic staining, typically High in bone marrow hematopoietic cells, lung macrophages or splenic red-pulp cells (HPA tissue IHC: High; cytoplasmic); strong staining of bronchial respiratory epithelium is suspect (HPA tissue IHC: Not detected).
| Cell population represented in the section | Interpret staining by cell type: bone marrow hematopoietic cells, lung macrophages and splenic red-pulp cells are High, while lymph-node non-germinal-center cells are Medium (HPA tissue IHC). A tissue-wide positive or negative label would obscure this distinction. |
| Compartment and protein topology | HPA reports cytoplasmic staining; UniProt annotates neither a transmembrane segment nor a signal peptide (HPA tissue IHC: cytoplasmic; UniProt P52790 topology and processing). A dominant membrane outline therefore needs independent scrutiny, though these annotations do not determine every possible intracellular distribution. |
| Strength of IHC evidence | The tissue pattern is rated Enhanced for consistency between antibody staining and RNA expression; the listed antibody HPA056743 also has Enhanced IHC status (HPA tissue IHC: reliability; HPA antibodies: HPA056743). These ratings support the reported pattern but do not validate every new section or staining run. |
| Limits of subcellular IF evidence | HPA provides no ICC-IF images or main subcellular location for HK3 (HPA subcellular ICC-IF: no images; location unavailable). Use the tissue IHC cytoplasmic pattern for this IHC guide; do not present a separate experimentally verified IF compartment from this record. |
| Situation | Likely cause | Next action |
|---|---|---|
| The positive control is blank. | Target-specific fixation effects are not established by the supplied assay evidence. Verify with a matched IHC source before attributing a result to fixation. | Confirm the control contains the expected cells, then review retrieval, antibody application and chromogenic detection using standard IHC controls (general IHC practice). |
| Most of the section has weak, diffuse color. | Background can obscure selective cytoplasmic staining (HPA tissue IHC: selective cytoplasmic expression). Nonspecific binding or detection background are general IHC possibilities (general IHC practice). | Compare the no-primary control; review blocking, washing and detection exposure before scoring individual cells (general IHC practice). |
| Brown deposit appears in cells expected to be negative. | The pattern conflicts with HPA's Not detected calls for bronchial respiratory epithelial cells and adipocytes; cross-reactivity or endogenous detection activity are possibilities, not established causes (HPA tissue IHC: bronchus; adipose tissue). | Verify cell identity and compare no-primary and detection controls; score the questionable population separately from nearby immune cells (general IHC practice; HPA tissue IHC: selective immune-cell expression). |
| Signal is mainly nuclear or outlines cell membranes. | That differs from the reported cytoplasmic pattern; UniProt lists no transmembrane segment (HPA tissue IHC: cytoplasmic; UniProt P52790 topology). | Inspect a known-positive cell population and controls, then reassess localization before assigning HK3 positivity (HPA tissue IHC: High in lung macrophages; general IHC practice). |
| A whole tissue is called negative despite scattered stained cells. | HK3 staining is selective by cell population, including splenic red-pulp cells and lymph-node non-germinal-center cells (HPA tissue IHC: High and Medium, respectively). | Identify and score the reported cell population rather than averaging its signal across the full section (HPA tissue IHC: cell-specific staining; general IHC practice). |
| Can an IF or ICC image confirm the precise HK3 compartment? | HPA lists no ICC-IF images and no main subcellular location for HK3 (HPA subcellular ICC-IF: images absent; location unavailable). | Treat any IF localization as a separate result requiring its own controls and guide; this section supports the reported tissue IHC cytoplasmic pattern (HPA tissue IHC: cytoplasmic; general IF practice). |
Experiment evidence
IHC & IF images showing HK3 staining
A05145-3 · IHCIHC analysis of HK3 using anti-HK3 antibody (A05145-3). HK3 was detected in a paraffin-embedded section of human liver tissue. Heat mediated
A05145-3 · IHCIHC analysis of HK3 using anti-HK3 antibody (A05145-3). HK3 was detected in a paraffin-embedded section of human liver tissue. Heat mediated
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000160883-HK3/tissue/cerebral+cortex#imid_17608563
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000160883-HK3/tissue/colon#imid_17611145
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000160883-HK3/tissue/liver#imid_17611138
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000160883-HK3/tissue/kidney#imid_17611147
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for HK3 IHC & IF
🧪Run lung first and require staining in macrophages (HPA: High in lung macrophages). Use bronchus respiratory epithelium as the negative tissue (HPA: Not detected in bronchus respiratory epithelial cells); where airway epithelium is present on the lung slide, use unstained epithelial cells as an internal negative comparison (HPA: Not detected in bronchus respiratory epithelial cells).
Positive control tissue: Bone marrow (Hematopoietic cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA carries no ICC-IF cell line for HK3; derive a cell-line control from the positive tissue's cell type (Hematopoietic cells) and confirm it by RNA or western blot first.
Technical controls: Include a no-primary, secondary-only control and a concentration-matched rabbit IgG isotype control (caption: rabbit anti-HK3 primary); use HK3 knockout material, if available, as a biological negative (standard IHC practice). Quench endogenous peroxidase and inspect macrophage pigment in lung sections (HPA: High in lung macrophages; standard IHC practice).
⚠️Feasibility: No target-specific fixation window or fixation effect is reported, and the selected A05145-3 paraffin-section caption does not state a fixative (caption: fixative not stated). Heat-mediated retrieval in EDTA at pH 8.0 is documented for that IHC example, but whether retrieval is required has not been established (caption: EDTA retrieval). The evidence does not establish that frozen sections or IF are easier (HPA: no ICC-IF images); lung macrophage pigment may complicate chromogenic scoring, and autofluorescence may complicate IF (HPA: High in lung macrophages; standard IHC/IF practice).
HPA tissue IHC evidence for HK3
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — High consistency between antibody staining and RNA expression data.). Rows are taken directly from the HPA tissue chart — click any row's HPA link to view the source.
Positive expression · recommended positive controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Bone marrow | Hematopoietic cells | High | Protein (IHC) | HPA → |
| Lung | Macrophages | High | Protein (IHC) | HPA → |
| Spleen | Cells in red pulp | High | Protein (IHC) | HPA → |
| Lymph node | Non-germinal center cells | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Adrenal gland | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Appendix | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Breast | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced HK3 IHC Tips
Troubleshoot HK3 staining in paraffin section IHC using the catalog antibody’s liver example and the reported immune cell staining pattern.
What should I adjust when HK3 staining is weak after retrieval?
Start with heat mediated antigen retrieval in EDTA pH 8.0 (datasheet A05145-3). The catalog antibody’s paraffin section example used that retrieval before applying 2 μg/ml primary antibody overnight at 4°C (datasheet A05145-3). If staining is weak, first confirm the bath reached its intended temperature, the sections stayed covered, and matched positive control sections were processed together (standard IHC practice). Then compare a modestly longer heating interval against the original interval while keeping buffer and detection conditions constant; inspect morphology and background before adopting the change (standard IHC practice).
Could fixation explain weak or uneven HK3 staining?
HK3 specific sensitivity to fixation is unknown because the selected paraffin section caption does not state a fixative (datasheet A05145-3). Record fixative, fixation duration, tissue thickness, and processing history for each specimen before comparing staining intensity (standard IHC practice). Run sections with different fixation histories through the same EDTA pH 8.0 retrieval and detection run, alongside a reference section, so a processing difference can be identified (datasheet A05145-3; standard IHC practice). If signal falls as fixation duration increases, treat that as an observation for those specimens and verify it with matched samples before changing the workflow (standard IHC practice).
Where should convincing HK3 staining appear in a tissue section?
Prioritise cytoplasmic staining in hematopoietic cells and subsets of immune cells when reading HK3 IHC (HPA: selective cytoplasmic expression). Strong reported examples include bone marrow hematopoietic cells and lung macrophages; spleen red pulp cells also stain strongly (HPA: tissue IHC). HK3 has no annotated transmembrane segment, while UniProt gives no specific subcellular location, so a sharply membranous pattern alone needs independent validation (UniProt P52790 topology and subcellular annotation). Compare positive cells with nearby cells in the same section and with a matched negative control, assessing staining distribution as well as intensity (standard IHC practice).
How can I assess whether unexpected staining reflects epitope recognition?
Check the catalog antibody’s stated immunogen or epitope before interpreting an unexpected compartment or cell population; its precise epitope is not supplied here (datasheet A05145-3: epitope not supplied). UniProt lists one 923 amino acid HK3 chain, no annotated isoforms, no signal peptide, and no annotated glycosylation sites or modified residues (UniProt P52790). Those annotations do not establish antibody specificity in a paraffin section (standard IHC interpretation). Compare staining with an independent antibody targeting a distinct known region, if available, and require agreement in cell type and cytoplasmic distribution rather than relying on staining intensity alone (HPA: selective cytoplasmic expression; standard IHC practice).
How should I investigate HK3 by IF alongside this IHC assay?
Treat IF as a separate assay requiring its own antibody validation; the selected product evidence describes chromogenic staining of a paraffin section (datasheet A05145-3). For multiplex IF, pair HK3 with a validated marker for the expected immune cell population, such as macrophages in lung, and include single stain controls (HPA: high in lung macrophages; standard IF practice). Choose spectrally separated fluorophores after checking tissue autofluorescence and image each channel under the same acquisition settings (standard IF practice). If testing intracellular access, optimise permeabilisation for a cytoplasmic target; no membrane sided epitope is specified for this antibody (HPA: selective cytoplasmic expression; datasheet A05145-3: epitope not supplied).
What causes diffuse brown signal in HK3 IHC?
Assess whether brown signal follows the expected cytoplasmic immune cell pattern or appears broadly in unrelated cells and extracellular regions (HPA: selective cytoplasmic expression; standard IHC practice). The selected assay uses a peroxidase linked secondary antibody and DAB, so a peroxidase block and a no primary control help distinguish detection background from antibody dependent staining (datasheet A05145-3; standard IHC practice). The example used 10% goat serum for blocking and 2 μg/ml primary antibody overnight at 4°C (datasheet A05145-3). If background persists, titrate the primary antibody and check washing and DAB development using adjacent sections (standard IHC practice).
How should I quantify HK3 across heterogeneous tissue sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and tissue area before scoring, because HK3 staining is selective among hematopoietic and immune cells (HPA: tissue IHC profile). For cell level comparisons, report the percentage of positive cells and an intensity based H-score within the same annotated cell population (standard IHC practice). For spatial comparisons, report positive cell density per mm² of viable tissue, with separately defined regions if immune cells cluster (standard IHC practice). Keep retrieval, chromogen development, imaging, and positivity thresholds consistent across sections, and normalise counts to the number of evaluable cells or the measured viable area as appropriate (standard IHC practice).
How can I distinguish genuine HK3 staining from artefact?
A credible HK3 result shows a reproducible cytoplasmic pattern in the expected cell population, with strong reference staining in bone marrow hematopoietic cells or lung macrophages (HPA: tissue IHC). Diffuse nuclear only or sharply membranous staining needs additional validation because HPA reports cytoplasmic expression and UniProt specifies no transmembrane segment (HPA: tissue IHC profile; UniProt P52790 topology). Compare suspicious signal with section edges, damaged or necrotic areas, and the no primary control to identify processing or detection artefacts (standard IHC practice). In DAB assays, investigate persistent signal after omitting primary antibody as possible endogenous peroxidase activity or other detection background (standard IHC practice).
Boster reagents
Best HK3 / Hexokinase-3 IHC Antibodies
A05145-3 is human-reactive and has an IHC image from a paraffin-embedded human liver section (catalog: Human reactivity; A05145-3 IHC caption).
Real IHC data
Anti-HK3 Antibody ®
Cat # A05145-3
A05145-3 lists human IHC among its applications (catalog: applications and reactivity). Its IHC image shows a paraffin-embedded human liver section; no IF image is supplied (A05145-3 IHC caption; catalog: IF images).
Which to pick: Choose A05145-3 for human paraffin-section IHC: its caption documents heat retrieval in EDTA at pH 8.0, and its listed IHC concentration is 2–5 μg/ml (A05145-3 IHC caption; datasheet: IHC dilution); the fixative is unreported (A05145-3 IHC caption). No SKU in this payload is listed for IF/ICC or another species, so neither use is supported here (catalog: applications and reactivity). A05145-3 has a rabbit host, while clonality is unreported (catalog: host and clone).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P52790 (HXK3_HUMAN, Hexokinase-3).
- Human Protein Atlas. HK3 tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. HK3 subcellular location (ICC-IF): Highest expression in THP-1: 10.5 nTPM.
- Human Protein Atlas. HK3 antibody validation summary (1 antibodies).
- Reliable hexokinase 3 protein detection in human cell lines and primary tissue. European journal of histochemistry : EJH 2025 — PMC11956552.
- High iodine promotes autoimmune thyroid disease by activating hexokinase 3 and inducing polarization of macrophages towards M1. Frontiers in immunology 2022 — PMC9618622.
- Differential proteomic analysis of lung tissues in rats with high-altitude pulmonary edema and screening of potential biomarkers. Frontiers in physiology 2026 — PMC13616702.
- Dual roles of HK3 in regulating the network between tumor cells and tumor-associated macrophages in neuroblastoma. Cancer immunology, immunotherapy : CII 2024 — PMC11076449.
- PubMed PMID:8812439 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
- PubMed PMID:8717435 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for HK3 (Hexokinase-3). Expected localisation Cytoplasmic staining in hematopoietic and immune cells (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A05145-3). Validated IHC antibody: A05145-3 at 2-5 μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Bone marrow (Hematopoietic cells); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human liver tissue; fixative not specified (datasheet A05145-3); 2. Fixation: Image fixative and duration unreported (datasheet A05145-3); 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 A05145-3); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A05145-3); 8. Primary antibody: Rabbit anti-HK3, 2-5 μg/ml (datasheet A05145-3); 9. Primary incubation: Overnight at 4 °C (datasheet A05145-3). Watch for: Fixation: Keep fixation consistent across sections. (standard IHC practice; not target-specific); Caveat: Immune-cell abundance can change apparent tissue signal (HPA tissue IHC); Isoform / epitope: One 1–923 chain; no isoforms or processing annotated (UniProt); Regulation: Cell-type-dependent expression (HPA tissue IHC). Evidence: UniProt P52790, HPA tissue IHC · Enhanced, HPA cell-line RNA · Highest expression in THP-1: 10.5 nTPM, Datasheet A05145-3, PMC11956552, PMC9618622. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/HK3/immunohistochemistry-ihc-guide.