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HK1 / Hexokinase-1 · IHC design guide
Design Immunohistochemistry for HK1
Plan HK1 paraffin IHC around granular cytoplasmic staining (HPA tissue IHC) and its molecular association with the mitochondrial outer membrane (UniProt). Neuronal cells in the cerebral cortex show high staining and can serve as a positive reference (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
HK1 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 | Observed granular cytoplasm (HPA tissue IHC); molecular outer mitochondrial membrane association (UniProt) | |
| Staining pattern | Ubiquitous granular cytoplasmic staining (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet M01504-2) | |
| Positive control | Appendix+4 more · see all | |
| Negative control | Adipose tissue+1 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 | Adipocytes may lack signal despite the broad tissue profile (HPA tissue IHC) | |
| Regulation | GlcNAc shifts HK1 to cytosol (UniProt) | |
| Isoform / epitope | 4 isoforms; 2 is erythrocyte-specific, 3–4 testis-specific; epitope coverage unknown (UniProt) |
Section 1
Recommended HK1 IHC & IF Protocols
The catalog antibody’s IHC-P protocol (datasheet: M01504-2) is accompanied by published chromogenic HK1 methods for mouse tissue sections and mink uterus (PMC7658079; PMC4284318).
| Sample | Paraffin-embedded human lung cancer tissue; fixative not specified (datasheet M01504-2) |
| Fixation | Image fixative and duration unreported (datasheet M01504-2); 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 M01504-2); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet M01504-2) |
| Primary antibody | Mouse monoclonal (clone 4B7) anti-HK1, 0.5-1μg/ml (datasheet M01504-2) |
| Primary incubation | Overnight at 4 °C (datasheet M01504-2) |
| Detection | Streptavidin-biotin complex (SABC), DAB chromogen (datasheet M01504-2) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | HK1-positive staining in endocrine cells of appendix (HPA tissue IHC: High). HPA tissue profile: Ubiquitous cytoplasmic expression with a granular pattern. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA at pH 8.0 (datasheet: M01504-2); the mink uterine method used sodium citrate at 90 °C for 20 min (PMC4284318).
Section 2
What Is the Expected HK1 Staining Pattern?
HK1 should appear as granular cytoplasmic staining in many cell types, with especially strong signal in selected endocrine, epithelial, and neuronal cells (HPA tissue IHC: Supported; ubiquitous granular cytoplasmic profile). Its mitochondrial outer membrane association and cytosolic pool explain the cytoplasmic location; HK1 has no transmembrane segment (UniProt P19367: subcellular location and topology). HPA reports medium agreement between antibody staining and RNA expression (HPA tissue IHC: reliability description).
| Granular cytoplasmic staining is strong in cerebral cortex neurons, bronchial respiratory epithelium, or appendix endocrine cells (HPA tissue IHC: High in these cells). | This matches the reported tissue pattern and is a useful positive-slide check (HPA tissue IHC: ubiquitous granular cytoplasmic profile; High in these cells). Judge the signal in the named cells, since a mixed tissue contains cells with different reported staining levels (HPA tissue IHC: cell-level annotations). |
| Signal appears mainly nuclear, with little cytoplasmic staining. | A predominantly nuclear result conflicts with the reported mitochondrial and cytosolic locations and granular cytoplasmic IHC pattern (UniProt P19367: subcellular location; HPA tissue IHC: profile). Treat it as suspect staining and review the counterstain, detection background, and control slide before calling HK1 positive (standard IHC practice). |
| Adipocytes or chondrocytes stain as strongly as the selected positive cells. | HPA reports HK1 as not detected in adipocytes and chondrocytes (HPA tissue IHC: Not detected). Unexpected staining raises concern for cross-reactivity, endogenous detection activity, or slide background; it does not identify which cause is responsible (standard IHC practice). Compare the pattern with a no-primary control (standard IHC practice). |
| Color spreads evenly across tissue and empty slide areas, obscuring cell outlines. | A uniform haze does not resemble HK1's reported granular cytoplasmic distribution (HPA tissue IHC: profile). Excess detection background or insufficient blocking or washing can obscure a genuine signal (standard IHC practice). Interpret intensity only after the background is brought under control (standard IHC practice). |
| No signal is visible in a section containing cerebral cortex neurons or esophageal squamous epithelium. | Both cell populations are reported High by HPA, so a blank result calls for an assay check (HPA tissue IHC: High in cerebral cortex neuronal cells and esophageal squamous epithelial cells). Verify that those cells are present, then review antibody, retrieval, and detection performance (standard IHC practice). A blank slide alone does not establish absent HK1. |
💡Expected HK1 appearanceCall a result positive when the named HPA High cells show clear, predominantly granular cytoplasmic staining above local background; mainly nuclear color or equally strong staining in HPA Not detected adipocytes or chondrocytes warrants investigation (HPA tissue IHC: profile and cell-level staining; UniProt P19367: subcellular location).
| Compartment and topology (UniProt P19367: subcellular location and topology) | HK1 associates with the mitochondrial outer membrane through a mitochondrial-binding region and can also reside in the cytosol; no transmembrane segment is annotated (UniProt P19367). Expect a cytoplasmic readout in chromogenic IHC, where individual mitochondria may not be resolved (HPA tissue IHC: granular cytoplasmic profile; standard IHC practice). |
| Cell choice for controls (HPA tissue IHC: cell-level staining) | Use a reported High population, such as hippocampal neurons or duodenal endocrine cells, when checking for a positive pattern (HPA tissue IHC: High). Adipocytes and chondrocytes are reported Not detected and can help flag unexpected color, but absence in them cannot by itself validate antibody specificity (HPA tissue IHC: Not detected; standard IHC practice). |
| Isoform coverage (UniProt P19367: isoforms and tissue specificity) | Four HK1 isoforms are listed; isoform 2 is erythrocyte specific, while isoforms 3 and 4 are testis specific (UniProt P19367). The supplied evidence does not locate the catalog antibody's epitope, so do not infer which isoforms it detects or use isoform specificity to explain a discrepant slide. |
| IHC evidence strength (HPA tissue IHC: Supported reliability; HPA antibodies: IHC Supported) | HPA describes medium consistency between antibody staining and RNA expression, and lists three antibodies with Supported IHC status (HPA tissue IHC: reliability description; HPA antibodies: IHC status). These observations support a pattern comparison, while an unexpected specimen still needs its own controls (standard IHC practice). |
| IF/ICC Q: What location should I expect? (HPA subcellular ICC-IF) | A: HPA reports mitochondrial localization as the enhanced main location in ICC-IF (HPA subcellular ICC-IF: main location). This helps interpret organelle-scale fluorescence; the chromogenic tissue-IHC expectation remains granular cytoplasm (HPA tissue IHC: profile). |
| Situation | Likely cause | Next action |
|---|---|---|
| The known-positive cell population is blank (HPA tissue IHC: High annotations). | The relevant cells may be missing from the section, or the retrieval, primary-antibody, or detection step may have failed (standard IHC practice). | Confirm cell identity on the counterstained section and run a reported High control beside the test section; review retrieval and reagent steps if both are blank (HPA tissue IHC: High annotations; standard IHC practice). |
| Granular signal is faint across a reported High population (HPA tissue IHC: profile and High annotations). | Weak assay signal or excessive background can make the expected cytoplasmic granules hard to distinguish (standard IHC practice). | Compare with the matched positive control and local background, then check antibody dilution and detection conditions against the available IHC procedure (standard IHC practice). Do not assign a new target-specific dilution from this evidence. |
| The slide shows widespread diffuse brown haze. | Nonspecific reagent retention or detection background can overwhelm cell-level staining (standard IHC practice). | Inspect a no-primary control; review blocking, washes, and detection exposure, then reassess whether granular cytoplasmic staining remains (HPA tissue IHC: profile; standard IHC practice). |
| Adipocytes or chondrocytes show conspicuous color (HPA tissue IHC: Not detected). | The finding conflicts with HPA's cell-level observations; cross-reactivity or endogenous detection activity are possibilities, not diagnoses (HPA tissue IHC: Not detected; standard IHC practice). | Compare a no-primary control and neighboring cell populations, and check endogenous enzyme blocking if an enzyme-based detection system is used (standard IHC practice). |
| Signal is predominantly nuclear. | That compartment differs from the reported mitochondrial and cytosolic locations (UniProt P19367: subcellular location). Counterstain or background may be mistaken for target signal (standard IHC practice). | Check the no-primary control and compare nuclear color with cytoplasmic granules in a reported High population before scoring the section (HPA tissue IHC: profile and High annotations; standard IHC practice). |
| One tissue looks weaker than another despite otherwise acceptable staining. | HK1 staining varies by cell population: HPA reports Low in liver cholangiocytes and High in hippocampal neurons (HPA tissue IHC: cell-level staining). | Score the specified cell type against its own background and an appropriate control. Do not treat lower intensity in a reported Low population as assay failure solely because a reported High population stains strongly (HPA tissue IHC: Low and High annotations; standard IHC practice). |
Experiment evidence
IHC & IF images showing HK1 staining
M01504-2 · IHCIHC analysis of Hexokinase 1/HK1 using anti-Hexokinase 1/HK1 antibody (M01504-2). Hexokinase 1/HK1 was detected in paraffin-embedded section
M01504-2 · IHCIHC analysis of Hexokinase 1/HK1 using anti-Hexokinase 1/HK1 antibody (M01504-2). Hexokinase 1/HK1 was detected in paraffin-embedded section
M01504-2 · IFIF analysis of Hexokinase 1/HK1 using anti-Hexokinase 1/HK1 antibody (M01504-2). Hexokinase 1/HK1 was detected in immunocytochemical section
A01504-1 · IHCIHC analysis of Hexokinase 1/HK1 using anti-Hexokinase 1/HK1 antibody (A01504-1). Hexokinase 1/HK1 was detected in paraffin-embedded section
A01504-1 · IHCIHC analysis of Hexokinase 1/HK1 using anti-Hexokinase 1/HK1 antibody (A01504-1). Hexokinase 1/HK1 was detected in paraffin-embedded section
A01504-1 · IFIF analysis of Hexokinase 1/HK1 using anti-Hexokinase 1/HK1 antibody (A01504-1). Hexokinase 1/HK1 was detected in immunocytochemical section
M01504-1 · IHCIHC analysis of Hexokinase 1/HK1 using anti-Hexokinase 1/HK1 antibody (M01504-1). Hexokinase 1/HK1 was detected in paraffin-embedded section
M01504-1 · IHCIHC analysis of Hexokinase 1/HK1 using anti-Hexokinase 1/HK1 antibody (M01504-1). Hexokinase 1/HK1 was detected in paraffin-embedded section
M01504-1 · IFIF analysis of Hexokinase 1/HK1 using anti-Hexokinase 1/HK1 antibody (M01504-1). Hexokinase 1/HK1 was detected in immunocytochemical section
A01504-2 · IHCImmunohistochemistry of Hexokinase 1 in rat stomach tissue with Hexokinase 1 antibody at 5 μg/mL.
A01504-2 · IFImmunofluorescence of Hexokinase 1 in rat stomach tissue with Hexokinase 1 antibody at 20 μg/mL.
M01504 · IHCImmunohistochemical analysis of paraffin-embedded human lung carcinoma, using Hexokinase 1 Antibody.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000156515-HK1/tissue/cerebral+cortex#imid_3315864
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000156515-HK1/tissue/colon#imid_3315806
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000156515-HK1/tissue/liver#imid_3315799
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000156515-HK1/tissue/kidney#imid_3315792
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody CAB010052; green = HK1, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody CAB010052; green = HK1, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for HK1 IHC & IF
🧪Run appendix first; its endocrine cells should stain strongly (HPA: High in appendix endocrine cells). Run adipose tissue as the negative comparator (HPA: Not detected in adipocytes); on the appendix slide, assess non-endocrine cells for background without assuming they are HK1-negative (HPA: only endocrine cells are specified as High).
Positive control tissue: Appendix (Endocrine cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show HK1 in A-431, U-251MG, U2OS, hTERT-RPE1 (serum starved), with annotated localisation: Mitochondria (enhanced) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control, a matched mouse IgG isotype control if the primary is monoclonal, and HK1-knockout material as a biological negative (caption: mouse anti-HK1; standard IHC controls). Block endogenous peroxidase and check endogenous biotin background in the appendix section when using the caption’s biotin–streptavidin/DAB detection (caption: biotinylated secondary, SABC and DAB; standard IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected M01504-2 tissue-IHC caption does not state a fixative (caption: fixative unreported). The paraffin-section example uses heat retrieval in EDTA at pH 8.0, but does not establish whether retrieval is required under other conditions (caption: EDTA pH 8.0). HPA shows mitochondrial ICC-IF localization, but the supplied evidence does not establish whether IF or frozen sections are easier; assess endogenous biotin and peroxidase background in appendix with the caption’s SABC/DAB method (HPA: mitochondria; caption: SABC/DAB; standard IHC practice).
HPA tissue IHC evidence for HK1
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Supported — Medium consistency between antibody staining and RNA expression data.). Rows are taken directly from the HPA tissue chart — click any row's HPA link to view the source.
Positive expression · recommended positive controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Appendix | Endocrine cells | High | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | High | Protein (IHC) | HPA → |
| Cerebellum | Cells in granular layer | High | Protein (IHC) | HPA → |
| Cerebral cortex | Neuronal cells | High | Protein (IHC) | HPA → |
| Colon | Endocrine cells | High | Protein (IHC) | HPA → |
Section 3
Advanced HK1 IHC Tips
Troubleshoot HK1 staining in paraffin sections by checking retrieval, controls and cell-level localisation before comparing signal across specimens.
What retrieval should I try first when HK1 staining is weak?
Start with heat-mediated antigen retrieval in EDTA at pH 8.0 (datasheet M01504-2). The catalog antibody produced HK1 staining in a paraffin section of human lung cancer tissue after that retrieval step (datasheet M01504-2). Keep heating, cooling and slide handling consistent across the test and control sections, then compare signal and tissue integrity (standard IHC practice). If staining remains weak, compare a second retrieval condition on adjacent sections while retaining EDTA pH 8.0 as the reference (standard IHC practice). Score granular cytoplasmic signal in expected cells rather than treating stronger diffuse colour as an automatic improvement (HPA tissue IHC: ubiquitous granular cytoplasmic expression).
Could fixation explain weak or uneven HK1 staining?
The selected paraffin-section caption does not report a fixative, so HK1-specific fixation sensitivity is unknown (datasheet M01504-2). Do not infer a fixation preference from the tissue staining pattern or HK1 membrane association (HPA tissue IHC; UniProt P19367 localisation). Compare sections with documented processing histories and include a similarly processed positive control when investigating weak signal (standard IHC practice). Check for uneven staining at section edges and within areas of poor preservation before changing antibody concentration (standard IHC practice). Record fixation details supplied with each specimen, because an unreported fixative cannot support a target-specific explanation for a failed stain (datasheet M01504-2; standard IHC practice).
Where should HK1 staining appear in a paraffin section?
Expect a granular cytoplasmic pattern in tissue IHC; the tissue profile describes that pattern as ubiquitous (HPA tissue IHC). HK1 associates with the mitochondrial outer membrane and can also occur in the cytosol, so both distributions are biologically plausible (UniProt P19367 localisation). Its mitochondrial-binding peptide promotes outer-membrane association, although that annotation is marked probable (UniProt P19367 localisation). Judge the pattern within intact cells against a positive control and the section’s morphology, since chromogenic granules alone cannot establish mitochondrial colocalisation (standard IHC practice). Predominantly nuclear or extracellular colour warrants a review of counterstain, background and tissue damage before biological interpretation (UniProt P19367 localisation; standard IHC practice).
Can this stain distinguish HK1 isoforms or phosphorylation states?
Do not assign an isoform from this chromogenic stain unless the antibody’s epitope and isoform coverage have been established (standard IHC practice). HK1 has 4 annotated isoforms; isoform 2 is erythrocyte specific, while isoforms 3 and 4 are testis specific (UniProt P19367 isoforms and tissue specificity). The record lists phosphoserine at residue 337, but the supplied antibody caption gives no phospho-specific recognition claim (UniProt P19367 modified residues; datasheet M01504-2). Check epitope documentation before comparing compartments or cell populations, and use an independently validated isoform-specific reagent if an isoform-level conclusion is essential (standard IHC practice). Report the observed signal as HK1 immunoreactivity until that specificity is demonstrated (standard IHC practice).
How should I assess HK1 localisation in a complementary IF experiment?
Use IF as a separate validation experiment and multiplex HK1 with a marker identifying the expected cell population (standard IF practice). Neuronal cells in cerebral cortex and respiratory epithelial cells in bronchus show high tissue-IHC staining, providing documented cell populations for that comparison (HPA tissue IHC). Choose a fluorophore channel with low tissue autofluorescence and include single-label controls before interpreting overlap (standard IF practice). HK1 associates with the mitochondrial outer membrane without an annotated transmembrane segment, while the antibody epitope is unspecified; optimise permeabilisation for antigen access and verify the observed compartment (UniProt P19367 topology and localisation; datasheet M01504-2). A mitochondrial marker can test spatial overlap, but overlap alone does not prove molecular interaction (standard IF practice).
How can I separate HK1 signal from chromogenic background?
Compare stained sections with a no-primary control to identify detection-system colour and tissue-associated background (standard IHC practice). The selected caption used 10% goat serum for blocking, a biotinylated goat anti-mouse secondary, streptavidin-biotin detection and DAB (datasheet M01504-2). Include an appropriate peroxidase block and inspect the no-primary control before attributing brown precipitate to HK1 (standard chromogenic IHC practice). If background persists, review secondary-only staining, blocking and development time on matched sections rather than interpreting diffuse colour as cytoplasmic expression (standard IHC practice). HK1’s reported tissue pattern is granular cytoplasmic, which gives a useful morphological check but cannot establish antibody specificity by itself (HPA tissue IHC; standard IHC practice).
What is a defensible way to quantify HK1 staining? ⚠ ANSWER MARKED FOR VERIFICATION
Define the scored cell population and tissue region before examining staining intensity, then apply the same criteria to every section (standard IHC practice). For chromogenic HK1 IHC, report an H-score combining the percentage of cells at each intensity, or report percentage-positive cells with a stated threshold (standard IHC practice). Normalise counts to the number of evaluable cells, or normalise positive-cell density to analysed area in mm² when cell counts are the outcome (standard IHC practice). Exclude folds, section edges and necrotic regions consistently, and keep illumination, colour separation and scoring settings fixed (standard IHC practice). Report cell type alongside scores because tissue expression varies by cell population (HPA tissue IHC).
When is an unexpected HK1 stain likely to be an artefact?
Treat granular cytoplasmic staining in intact cells as the pattern to assess first, because that is the reported tissue-IHC profile (HPA tissue IHC). High staining in bronchial respiratory epithelial cells or cerebral cortical neuronal cells is consistent with the reported cell-level profile; adipocytes and chondrocytes are listed as not detected (HPA tissue IHC). A dominant nuclear pattern conflicts with HK1’s annotated mitochondrial outer-membrane and cytosolic localisation and should prompt control review (UniProt P19367 localisation). Colour concentrated at edges, in necrosis or in the no-primary control suggests section or detection artefact; check peroxidase blocking and reagent controls (standard IHC practice). Tissue-IHC reliability is supported with medium RNA-staining consistency, so morphology and controls remain essential (HPA tissue IHC).
Boster reagents
Best HK1 / Hexokinase-1 IHC Antibodies
Anti-HK1 antibodies have IHC images from human lung tumors, mouse brain, and rat stomach, plus IF images from cells and tissue (catalog image captions).
Real IHC data
Anti-Hexokinase 1/HK1 Antibody ® (monoclonal, 4B7)
Cat # M01504-2
Real IHC data
Anti-Hexokinase 1/HK1 Antibody ®
Cat # A01504-1
Real IHC data
Anti-Hexokinase 1/HK1 Antibody ® (monoclonal, 2I4)
Cat # M01504-1
Real IHC data
Anti-Hexokinase 1 HK1 Antibody
Cat # A01504-2
Real IHC data
Anti-Hexokinase 1 HK1 Rabbit Monoclonal Antibody
Cat # M01504
M01504-2 has IHC images in human lung cancer and mouse intestine; A01504-1 in mouse and rat brain and human mammary and rectal cancers; M01504-1 in human lung, mammary, and testis cancers and frozen placenta (catalog IHC captions). A01504-2 has an IHC image in rat stomach, and M01504 in paraffin-embedded human lung carcinoma (catalog IHC captions).
Which to pick: For tissue IHC, start with M01504-2, a mouse monoclonal (4B7) with paraffin-section images in human lung cancer and mouse intestine and an IHC dilution of 0.5–1 μg/mL; its fixative is unreported (catalog host, clone, IHC captions and dilution). For IF/ICC, M01504-2 lists both applications and has IF images in U20S cells and human lung cancer sections at 2 μg/mL (catalog applications, IF captions and dilution). For cross-species IHC, A01504-1 lists human, mouse and rat reactivity and has paraffin-section images from all three species; its fixative is unreported (catalog reactivity and IHC captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P19367 (HXK1_HUMAN, Hexokinase-1).
- Human Protein Atlas. HK1 tissue IHC expression (reliability: Supported).
- Human Protein Atlas. HK1 subcellular location (ICC-IF): Localized to the mitochondria..
- Human Protein Atlas. HK1 antibody validation summary (3 antibodies).
- Direct neuronal glucose uptake heralds activity-dependent increases in cerebral metabolism. Nature communications 2015 — PMC4410436.
- Role of human tissue kallikrein in gastrointestinal stromal tumour invasion. British journal of cancer 2010 — PMC2990601.
- TAB1 regulates glycolysis and activation of macrophages in diabetic nephropathy. Inflammation research : official journal of the European Histamine Research Society ... [et al.] 2020 — PMC7658079.
- Uterine glycogen metabolism in mink during estrus, embryonic diapause and pregnancy. The Journal of reproduction and development 2014 — PMC4284318.
- PubMed PMID:3207429 — UniProt-cited evidence.
- PubMed PMID:9531504 — UniProt-cited evidence.
- PubMed PMID:15164054 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for HK1 (Hexokinase-1). Expected localisation Observed granular cytoplasm (HPA tissue IHC); molecular outer mitochondrial membrane association (UniProt). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet M01504-2). Validated IHC antibody: M01504-2 at 0.5-1μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Appendix (Endocrine cells); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human lung cancer tissue; fixative not specified (datasheet M01504-2); 2. Fixation: Image fixative and duration unreported (datasheet M01504-2); 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 M01504-2); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet M01504-2); 8. Primary antibody: Mouse monoclonal (clone 4B7) anti-HK1, 0.5-1μg/ml (datasheet M01504-2); 9. Primary incubation: Overnight at 4 °C (datasheet M01504-2). Watch for: Fixation: Keep fixation conditions consistent across sections. (standard IHC practice; not target-specific); Caveat: Adipocytes may lack signal despite the broad tissue profile (HPA tissue IHC); Isoform / epitope: 4 isoforms; 2 is erythrocyte-specific, 3–4 testis-specific; epitope coverage unknown (UniProt); Regulation: GlcNAc shifts HK1 to cytosol (UniProt). Evidence: UniProt P19367, HPA tissue IHC · Supported, HPA ICC-IF · mitochondria., Datasheet M01504-2, PMC7658079, PMC4284318. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/HK1/immunohistochemistry-ihc-guide.