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CHKB / Choline/ethanolamine kinase · IHC design guide
Design Immunohistochemistry for CHKB
Plan CHKB staining in paraffin sections around the general cytoplasmic pattern observed in tissue (HPA tissue IHC). Pancreatic exocrine glandular cells show high staining (HPA tissue IHC); the IHC-validated antibody A06297 has a stated dilution range of 1:50–1:200 (datasheet: A06297).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
CHKB 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 | General cytoplasmic tissue staining (HPA tissue IHC) | |
| Staining pattern | Cytoplasmic staining across multiple cell types (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen) | |
| Positive control | Pancreas+4 more · see all | |
| Negative control | None in HPA (detected in all 45 tissues); use no-primary + isotype controls |
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 | IHC staining and RNA show medium consistency (HPA tissue IHC) | |
| Regulation | No specific expression regulator reported (UniProt) | |
| Isoform / epitope | 2 isoforms; epitope differences are unreported (UniProt) |
Section 1
Recommended CHKB IHC & IF Protocols
The catalog antibody’s IHC-P protocol is accompanied by a published CHKB protocol for paraffin colon sections (PMC9701742).
| Sample | Paraffin-embedded human colorectal carcinoma tissue; fixative not specified (datasheet A06297) |
| Fixation | Image fixative and duration unreported (datasheet A06297); 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-CHKB, 1:50-1:200 (datasheet A06297) |
| 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 | CHKB-positive staining in exocrine glandular cells of pancreas (HPA tissue IHC: High). HPA tissue profile: General cytoplasmic expression. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6.0 HIER at 95–98 °C for 20 min (page retrieval rule); the published colon protocol also uses citrate (PMC9701742).
Section 2
What Is the Expected CHKB Staining Pattern?
CHKB should show predominantly cytoplasmic staining in tissue sections (HPA: general cytoplasmic expression). Stronger staining is reported in pancreatic exocrine glandular cells, placental decidual cells, smooth muscle cells, thyroid glandular cells and tonsillar non-germinal center cells (HPA: High). HPA rates its tissue IHC profile Approved, with medium consistency between staining and RNA data (HPA: reliability description). UniProt annotates no transmembrane segment and no subcellular location (UniProt Q9Y259 topology and subcellular record).
| Cytoplasmic chromogen in the expected cells, with stronger signal in a high-staining reference tissue (HPA: general cytoplasmic expression; High in pancreatic exocrine glandular cells). | This fits the reported tissue pattern. Score the relevant cells and their cytoplasm, then compare intensity with surrounding tissue on the same section (HPA: tissue IHC profile; general IHC practice). HPA's Approved rating has medium staining–RNA consistency, so morphology and controls remain important (HPA: reliability description). |
| Predominantly nuclear, membranous or extracellular signal, with little cytoplasmic staining. | That distribution conflicts with the reported general cytoplasmic IHC pattern and approved cytosolic ICC-IF location (HPA: tissue IHC; HPA: ICC-IF). Treat it as a possible artefact and review morphology and controls (general IHC practice). UniProt lists no subcellular annotation; its lack of a transmembrane segment alone cannot prove the compartment (UniProt Q9Y259). |
| Strong staining chiefly in cells outside the expected population, while the expected cells are weak. | Check cell identity before calling this a different CHKB pattern (general IHC practice). Unexpected staining can reflect antibody cross-reactivity or endogenous detection activity; neither is established by appearance alone (general IHC practice). HPA reports low tissue specificity, so staining beyond its listed high-staining cells is possible (HPA: RNA specificity). |
| Diffuse chromogen over cells and surrounding tissue, with poor distinction between cytoplasm and background. | The reported result is general cytoplasmic expression, so an indiscriminate deposit is difficult to score as CHKB (HPA: tissue IHC profile). Compare a reagent control and inspect wash, blocking and detection steps for nonspecific signal (general IHC practice); the pattern alone does not identify its cause. |
| No convincing cytoplasmic signal in a reference section expected to stain strongly. | First confirm that the relevant cell population is present: HPA reports High staining in pancreatic exocrine glandular cells, among other listed cells (HPA: tissue IHC). A blank result may reflect an assay problem, but HPA supplies no target-specific fixation sensitivity or guaranteed positive result for every specimen (HPA: tissue IHC scope). |
💡Expected CHKB appearanceCall a result supportive when identifiable cells show predominantly cytoplasmic chromogen, strongest in an HPA High population such as pancreatic exocrine glandular cells; dominant nuclear, membranous or diffuse extracellular deposit is suspect (HPA: tissue IHC; HPA: ICC-IF; general IHC practice).
| Reference cell population and intensity | HPA rates five listed populations High, including pancreatic exocrine glandular and smooth muscle cells; adrenal glandular, appendix glandular and marrow hematopoietic cells are Medium (HPA: tissue IHC). Select and score the actual reported cell population, since a tissue name alone does not specify where signal should appear (general IHC practice). |
| Lower-staining comparators | HPA lists cerebellar granular-layer cells, esophageal and vaginal squamous cells, peripheral nerve in soft tissue, and adipocytes as Low (HPA: tissue IHC). Low means less reported staining, not a validated absence of CHKB; HPA supplies no negative tissue in this payload (HPA: Low and negative lists). |
| Compartment and assay scope | Tissue IHC is described as generally cytoplasmic, while ICC-IF independently reports an approved cytosolic location (HPA: tissue IHC; HPA: ICC-IF). UniProt has no subcellular annotation and lists no transmembrane segment (UniProt Q9Y259), so avoid inferring a more precise tissue distribution from topology. |
| Antibody validation and isoforms | The listed rabbit polyclonal antibody HPA018797 is Approved for IHC and ICC, but this record does not report an IHC Enhanced rating (HPA: antibody validation). UniProt lists two CHKB isoforms; the payload gives no epitope or isoform-reactivity information, so do not assign a staining difference to either isoform (UniProt Q9Y259 isoforms). |
| IF/ICC Q: what location is expected? | A: Cytosolic signal is the reported expectation (HPA: ICC-IF, Cytosol approved). This ICC-IF observation supports a compartment check, but it does not establish the intensity of chromogenic staining in paraffin tissue sections (HPA: ICC-IF; HPA: tissue IHC). |
| Situation | Likely cause | Next action |
|---|---|---|
| A high-staining reference section appears blank. | The expected cell population may be absent, or an IHC step may have failed (HPA: High cell populations; general IHC practice). | Confirm morphology and the listed cell type, then check primary antibody, retrieval and detection against the validated IHC procedure (general IHC practice). HPA provides no CHKB-specific retrieval condition or fixation-effect claim (HPA: tissue IHC scope). |
| Signal is concentrated in nuclei or along cell borders. | The compartment differs from HPA's general cytoplasmic tissue pattern and approved cytosolic ICC-IF location (HPA: tissue IHC; HPA: ICC-IF). | Inspect counterstain, section morphology and reagent controls; repeat with the IHC-validated antibody if the pattern persists (general IHC practice; HPA: IHC Approved). Do not score compartment-mismatched signal as confirmed CHKB. |
| The entire section has a diffuse chromogenic haze. | Nonspecific reagent binding or endogenous detection activity can obscure a cell-specific signal (general IHC practice). | Review blocking, washing and detection controls, and compare the reagent control with the test section (general IHC practice). Score CHKB only where a distinguishable cytoplasmic pattern remains (HPA: general cytoplasmic expression). |
| An unexpected cell type is much stronger than the listed reference cells. | Cell identification, cross-reactivity or endogenous detection activity may explain the mismatch (general IHC practice); HPA also reports low tissue specificity (HPA: RNA specificity). | Verify the cell type morphologically and compare staining with the matching HPA cell-level profile and controls (HPA: tissue IHC; general IHC practice). Treat an unexplained mismatch as uncertain rather than proof of a new expression pattern. |
| A low-staining comparator shows faint cytoplasmic signal. | Low is the reported staining level for several cell populations, including adipocytes and esophageal squamous cells (HPA: Low); it is not a negative-control designation. | Record the cell type and relative intensity, then compare with the high-staining reference and reagent control (HPA: tissue IHC; general IHC practice). Do not classify faint signal as failure solely because the comparator is listed Low. |
| ICC-IF appears cytosolic, but paraffin IHC is inconclusive. | The assays have separate HPA observations: approved cytosolic ICC-IF and general cytoplasmic tissue IHC (HPA: ICC-IF; HPA: tissue IHC). | Interpret the IHC section using its own morphology, reference cells and controls (general IHC practice). Use the ICC-IF result as a compartment cross-check, without treating it as an IHC protocol or an intensity benchmark (HPA: ICC-IF; HPA: tissue IHC). |
Experiment evidence
IHC & IF images showing CHKB staining
A06297 · IHCImmunohistochemistry (IHC) analyzes of ChoKB (E324) pAb in paraffin-embedded human colorectal carcinoma tissue at 1:50.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000100288-CHKB/tissue/cerebral+cortex#imid_4864712
HPA · Thyroid glandHuman Protein Atlas IHC, Thyroid gland. Source: https://www.proteinatlas.org/ENSG00000100288-CHKB/tissue/thyroid+gland#imid_4864656
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000100288-CHKB/tissue/colon#imid_4864630
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000100288-CHKB/tissue/liver#imid_4864647
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA018797; green = CHKB, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA018797; green = CHKB, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for CHKB IHC & IF
🧪Run pancreas first and look for staining in exocrine glandular cells (HPA: High in pancreas exocrine glandular cells). HPA detects CHKB in all 45 scored tissues, so use no-primary and isotype controls for the negative comparison; on the positive slide, count a cell as an internal negative only if it shows no specific chromogen above background, without assuming any cell type is CHKB-negative (HPA: no negative tissue listed; standard IHC practice).
Positive control tissue: Pancreas (Exocrine glandular cells, HPA High)
Negative control tissue: None in HPA: CHKB is detected in all 45 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 CHKB in A-431, U-251MG, U2OS, with annotated localisation: Cytosol (approved) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control, an isotype-matched control appropriate to the primary antibody’s host and clonality, and a CHKB-knockout specimen if available (standard IHC practice). For pancreas, quench endogenous peroxidase and check endogenous biotin background if using avidin-biotin detection (standard IHC practice).
⚠️Feasibility: No matched source reports a CHKB-specific fixation window or antigen-retrieval dependence; the selected A06297 paraffin-section caption does not state a fixative (selected product caption). Paraffin-section IHC is documented at 1:50 in human colorectal carcinoma, but that caption does not establish fixation (selected product caption). HPA ICC-IF images support cytosolic localization in A-431, U-251MG and U2OS cells, but the supplied evidence does not establish that frozen sections or IF are easier; in pancreas, assess peroxidase and, for avidin-biotin detection, biotin background (HPA: subcellular; standard IHC practice). The selected A06297 tissue-IHC caption documents paraffin sections, but does not specify the fixative (selected-SKU IHC image A06297).
HPA tissue IHC evidence for CHKB
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — 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 |
|---|---|---|---|---|
| Pancreas | Exocrine glandular cells | High | Protein (IHC) | HPA → |
| Placenta | Decidual cells | High | Protein (IHC) | HPA → |
| Smooth muscle | Smooth muscle cells | High | Protein (IHC) | HPA → |
| Thyroid gland | Glandular cells | High | Protein (IHC) | HPA → |
| Tonsil | Non-germinal center cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| None in HPA: CHKB is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead. | ||||
Section 3
Advanced CHKB IHC Tips
Troubleshoot CHKB staining in paraffin sections using the page retrieval method, the catalog image, and independent localisation evidence.
What retrieval should I try first when CHKB staining is weak?
Use citrate pH 6.0 heat-induced epitope retrieval at 95–98 °C for 20 min first (page retrieval rule). Allow sections to cool consistently, then compare staining with a positive control section processed in the same run (standard IHC practice; HPA: high staining in pancreatic exocrine glandular cells). The A06297 image shows staining in paraffin-embedded human colorectal carcinoma at 1:50, but its caption gives no retrieval method (A06297 caption). If staining remains weak, test an alternative retrieval condition on adjacent sections while keeping antibody dilution and detection constant (standard IHC practice). Judge improvement by clearer cellular staining without a parallel rise in tissue-wide background (standard IHC practice).
Could fixation explain variable CHKB staining between paraffin blocks?
CHKB-specific fixation sensitivity is unknown because no target-specific fixation experiment is supplied (evidence payload). The A06297 tissue image is described as paraffin-embedded human colorectal carcinoma, with no fixative stated, so it cannot establish a preferred fixative or fixation duration (A06297 caption). Record each block's fixative and processing history, and compare blocks using the same citrate pH 6.0, 95–98 °C, 20 min retrieval and detection conditions (page retrieval rule; standard IHC practice). Include a control with expected cytoplasmic staining in every run, such as pancreatic exocrine glandular cells (HPA: high staining in pancreatic exocrine glandular cells). Interpret block-to-block differences cautiously when processing histories differ (standard IHC practice).
Where should convincing CHKB chromogenic staining appear?
Expect predominantly cytoplasmic staining: tissue IHC is described as generally cytoplasmic, and independent subcellular data place CHKB in the cytosol (HPA tissue: general cytoplasmic expression; HPA subcellular: cytosol, approved). UniProt reports no transmembrane segment and does not annotate a subcellular location, so topology alone cannot establish the staining pattern (UniProt Q9Y259 topology and subcellular record). Compare signal within identified cells against adjacent background using the same counterstain and exposure settings (standard IHC practice). Strong nuclear-only or sharply membranous staining should prompt review of morphology, controls, and detection background before it is assigned to CHKB (HPA subcellular: cytosol; standard IHC practice). Cytoplasmic intensity can vary by cell population (HPA tissue IHC profile).
Can this antibody distinguish CHKB isoforms in tissue?
CHKB has 2 listed isoforms, but the supplied antibody caption does not map its epitope to either one (UniProt Q9Y259 isoforms; A06297 caption). Do not interpret a positive or negative chromogenic signal as isoform-specific without an epitope sequence and independent isoform controls (standard IHC interpretation). The record describes a 395 aa protein with no transmembrane segment and no annotated glycosylation sites; those facts do not reveal which residues the antibody recognizes (UniProt Q9Y259 length, topology, and glycosylation). Compare adjacent sections under one retrieval condition and document the antibody identity when assessing discordant staining (standard IHC practice). An alternative antibody with a mapped, nonoverlapping epitope can help investigate a disputed pattern (standard IHC practice).
How should I assess CHKB in a tissue IF multiplex panel?
Use the separate IF/ICC guide for its application-specific workflow; this page's A06297 image establishes only paraffin-section IHC at 1:50 (A06297 caption). In a multiplex experiment, pair CHKB with a marker identifying the cell population being assessed, such as a validated glandular-cell marker when evaluating pancreatic exocrine cells (HPA: high staining in pancreatic exocrine glandular cells; standard IF practice). Choose fluorophores after inspecting unstained tissue for autofluorescence, and place the weaker signal in a channel with low tissue background (standard IF practice). Because the expected signal is cytosolic and CHKB has no transmembrane segment, assess whether permeabilisation permits access to intracellular epitopes while preserving cell boundaries (HPA subcellular: cytosol; UniProt Q9Y259 topology; standard IF practice).
How can I separate CHKB staining from chromogenic background?
Examine a no-primary control alongside the CHKB-stained section to identify signal from the detection reagents or tissue itself (standard IHC practice). Include a peroxidase block before an enzyme-based DAB workflow and inspect whether brown deposits persist in the control (standard IHC practice). Titrate the catalog antibody from the caption's 1:50 condition on matched sections while keeping retrieval at citrate pH 6.0, 95–98 °C, 20 min (A06297 caption; page retrieval rule; standard IHC practice). Prefer a condition that retains defined cytoplasmic staining without diffuse stromal or edge staining (HPA tissue: general cytoplasmic expression; standard IHC practice). HPA lists low staining in several cell populations, which can aid comparison but does not make them validated negative controls (HPA tissue IHC profile).
How should CHKB IHC be scored across samples? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and viable tissue area before scoring, then apply identical retrieval, detection, counterstain, and image settings across samples (standard IHC practice). For cytoplasmic CHKB, report the percentage of positive target cells and an intensity-based H-score, with thresholds set from controls before comparing groups (HPA tissue: general cytoplasmic expression; standard IHC practice). If counting positive cells, express density per mm² of viable, evaluable tissue rather than total section area (standard IHC practice). Report scores separately by cell population because HPA records high staining in pancreatic exocrine glandular cells and decidual cells, among others (HPA tissue IHC profile). Record excluded necrotic and damaged regions so differences in tissue quality do not drive the comparison (standard IHC practice).
What would make a CHKB-positive result convincing?
Look for reproducible cytoplasmic signal within intact cells, supported by an appropriate positive control and minimal staining in a no-primary control (HPA tissue: general cytoplasmic expression; standard IHC practice). Pancreatic exocrine glandular cells are one reported high-staining population, but staining elsewhere requires evaluation in its own cellular context (HPA: high staining in pancreatic exocrine glandular cells; HPA tissue: low tissue specificity). Treat nuclear-only signal, staining confined to section edges or necrosis, and deposits also present in the no-primary control as possible artefacts (HPA subcellular: cytosol; standard IHC practice). Check endogenous peroxidase contribution when using DAB, and compare the suspect region with adjacent intact tissue (standard IHC practice). HPA rates tissue staining as Approved with medium consistency against RNA expression, so staining alone should not establish biological specificity (HPA tissue reliability).
Boster reagents
Best CHKB / Choline/ethanolamine kinase IHC Antibodies
Anti-CHKB A06297 has an IHC image from a human paraffin-embedded colorectal carcinoma section (catalog image caption); both antibodies list human, mouse and rat reactivity (catalog applications/reactivity). No IF image is supplied (catalog images).
Real IHC data
Anti-ChoKB (E324) CHKB Antibody
Cat # A06297
A06297 will render with its own IHC image of human paraffin-embedded colorectal carcinoma at 1:50 (catalog image caption). Its listed application is IHC, with human, mouse and rat reactivity; the fixative is unreported (catalog applications/reactivity; catalog image caption).
Which to pick: Choose A06297 for tissue IHC when an imaged paraffin-section example matters; its caption documents human colorectal carcinoma at 1:50, but does not report the fixative (catalog image caption). For IF/ICC, A06297-1 lists IF and IHC, though no IF image or ICC validation is supplied (catalog applications/images). Both list human, mouse and rat reactivity, so neither has a documented cross-species advantage; A06297 is described as pAb in its image caption, while A06297-1 has no reported clone designation (catalog reactivity; catalog image caption; catalog clone field).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q9Y259 (CHKB_HUMAN, Choline/ethanolamine kinase).
- Human Protein Atlas. CHKB tissue IHC expression (reliability: Approved).
- Human Protein Atlas. CHKB subcellular location (ICC-IF): Localized to the cytosol..
- Human Protein Atlas. CHKB antibody validation summary (1 antibodies).
- Construction of a novel choline metabolism-related signature to predict prognosis, immune landscape, and chemotherapy response in colon adenocarcinoma. Frontiers in immunology 2022 — PMC9701742.
- PubMed PMID:10918069 — UniProt-cited evidence.
- PubMed PMID:15461802 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for CHKB (Choline/ethanolamine kinase). Expected localisation General cytoplasmic tissue staining (HPA tissue IHC). Antigen retrieval: Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen). Validated IHC antibody: A06297 at 1:50-1:200 (the product's own IHC validation image is shown). Controls (HPA): positive Pancreas (Exocrine glandular cells); negative None in HPA (detected in all 45 tissues); use no-primary + isotype controls. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human colorectal carcinoma tissue; fixative not specified (datasheet A06297); 2. Fixation: Image fixative and duration unreported (datasheet A06297); 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-CHKB, 1:50-1:200 (datasheet A06297); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation conditions consistent across sections. (standard IHC practice; not target-specific); Caveat: IHC staining and RNA show medium consistency (HPA tissue IHC); Isoform / epitope: 2 isoforms; epitope differences are unreported (UniProt); Regulation: No specific expression regulator reported (UniProt). Evidence: UniProt Q9Y259, HPA tissue IHC · Approved, HPA ICC-IF · cytosol., Datasheet A06297, PMC9701742. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/CHKB/immunohistochemistry-ihc-guide.