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CHKB / Choline/ethanolamine kinase · IHC design guide
Design Immunohistochemistry for CHKB
Plan CHKB staining in paraffin sections using the general cytoplasmic pattern reported in tissue IHC (HPA tissue IHC). Pancreatic exocrine cells show high staining (HPA tissue IHC); titrate the IHC-validated antibody within 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 staining (HPA tissue IHC) | |
| Staining pattern | Cytoplasmic staining in pancreatic exocrine cells (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 consistent across paraffin sections. (standard IHC practice; not target-specific) | |
| Caveat | Antibody staining and RNA show medium consistency (HPA tissue IHC) | |
| Regulation | Staining-linked regulation is not reported (UniProt) | |
| Isoform / epitope | 2 isoforms; epitope coverage is unspecified (UniProt) |
Section 1
Recommended CHKB IHC & IF Protocols
Compare the catalog antibody’s IHC-P protocol (datasheet) with a published CHKB protocol for human and mouse colon paraffin 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 retrieval at 95–98 °C for 20 min (page retrieval rule); the article specifies citrate without those settings (PMC9701742).
Section 2
What Is the Expected CHKB Staining Pattern?
CHKB staining should be predominantly cytoplasmic in paraffin sections (HPA tissue IHC: general cytoplasmic expression). A cytosolic location is also observed by ICC-IF (HPA subcellular: approved cytosol); CHKB has no annotated transmembrane segment (UniProt Q9Y259 topology). Expect stronger staining in pancreatic exocrine glandular cells, placental decidual cells, smooth muscle cells, thyroid glandular cells, and tonsillar non-germinal center cells (HPA tissue IHC: High). The tissue IHC assessment is Approved, with medium consistency between staining and RNA data (HPA tissue IHC).
| Cytoplasmic staining is prominent in pancreatic exocrine glandular cells or thyroid glandular cells. | This fits reported High staining in those cell populations (HPA tissue IHC: High) and the reported cytoplasmic pattern (HPA tissue IHC). Compare the stained cells with tissue morphology before scoring intensity (general IHC practice). |
| Staining is mainly nuclear or sharply restricted to cell membranes. | That compartment differs from the reported cytoplasmic tissue pattern and approved cytosolic ICC-IF location (HPA tissue IHC; HPA subcellular). Treat it as a specificity or detection concern; morphology alone cannot identify its cause (general IHC practice). |
| The darkest signal is confined to a cell type other than the reported High populations. | Check cell identity and tissue context first: CHKB has low RNA tissue specificity, and HPA reports staining in several cell types (HPA tissue IHC). If the pattern remains discordant, assess possible antibody cross-reactivity or endogenous detection activity with appropriate controls (general IHC practice). |
| Chromogen covers multiple compartments or blank regions without clear cellular boundaries. | This diffuse background cannot establish CHKB localization (general IHC practice). Review blocking, reagent carryover, wash quality, and the detection-only control before interpreting faint cytoplasmic color (general IHC practice). |
| A known-positive region shows no visible staining. | Confirm that the section actually contains the reported positive cells; pancreas exocrine glandular cells and smooth muscle cells are High in HPA tissue IHC (HPA tissue IHC: High). If present, check assay controls and detection steps before calling CHKB absent (general IHC practice). |
💡Expected CHKB appearanceCall a result consistent with CHKB when cytoplasmic staining is clear in the reported High cell populations (HPA tissue IHC: High; general cytoplasmic expression); dominant nuclear or membrane-only color is discordant (HPA subcellular: approved cytosol).
| Tissue and cell selection | Use named cell populations when judging intensity: pancreatic exocrine glandular, placental decidual, smooth muscle, thyroid glandular, and tonsillar non-germinal center cells are High; adrenal glandular and bone marrow hematopoietic cells are Medium (HPA tissue IHC). HPA lists no negative tissue in this payload (HPA tissue IHC). |
| Strength of pattern evidence | The tissue IHC assessment is Approved but has medium consistency between staining and RNA expression (HPA tissue IHC). Treat a matching slide as supportive evidence, especially when cell identity and controls agree; it is not proof that every stained cell expresses CHKB specifically (general IHC practice). |
| Molecular form and antibody recognition | CHKB has two annotated isoforms and no annotated signal peptide or transmembrane segment (UniProt Q9Y259). The supplied record gives no epitope or isoform-specific antibody recognition data, so the expected stain cannot be assigned to one isoform (UniProt Q9Y259; HPA antibody record). |
| Does IF/ICC change the IHC expectation? | ICC-IF reports approved cytosolic localization in A-431, U-251MG, and U2OS cells (HPA subcellular). This supports a cytoplasmic interpretation of tissue IHC; the separate cell-imaging result supplies no paraffin-section staining intensity or IHC protocol conditions (HPA subcellular; HPA tissue IHC). |
| Chromogenic detection | Endogenous enzyme activity or detection-reagent binding can imitate cellular color in chromogenic IHC (general IHC practice). Use the detection-only control and the chemistry's appropriate blocking step to assess that possibility; the payload reports no CHKB-specific endogenous-activity effect (HPA tissue IHC). |
| Situation | Likely cause | Next action |
|---|---|---|
| No signal in pancreatic exocrine glandular cells. | The expected High population may be missing from the evaluated area (HPA tissue IHC: High), or an assay step may have failed (general IHC practice). | Verify morphology and a working positive control, then review the catalog antibody's IHC-P instructions and detection reagents (general IHC practice). |
| Weak staining in a reported High population. | Intensity may reflect tissue sampling or assay conditions; HPA's High label does not define the intensity of every section (HPA tissue IHC; general IHC practice). | Compare matching cell types and controls on the same run; review antibody dilution and antigen retrieval under the validated IHC-P procedure (general IHC practice). |
| Strong nuclear or membrane-only signal. | The compartment conflicts with reported cytoplasmic tissue staining and cytosolic ICC-IF localization (HPA tissue IHC; HPA subcellular). | Check localization against counterstain and cell boundaries; inspect controls and reassess antibody specificity before scoring the signal as CHKB (general IHC practice). |
| Color appears across blank spaces or many unrelated structures. | Diffuse background or detection chemistry may obscure a cellular pattern (general IHC practice). | Review washes, blocking, and detection-only controls; score CHKB only where cell-associated cytoplasmic staining can be distinguished (general IHC practice; HPA tissue IHC). |
| A low-staining cell type appears darker than the reported High cells. | HPA reports low staining in adipocytes and esophageal squamous epithelial cells, versus High staining in several named populations (HPA tissue IHC). | Recheck cell identification, compare an appropriate High reference tissue, and investigate detection background or cross-reactivity if the contrast persists (general IHC practice). |
| Adjacent samples give inconsistent positive-cell counts. | Different proportions of reported positive cell types can change the apparent tissue-wide result (HPA tissue IHC; general IHC practice). | Score the named cell population and its cytoplasmic pattern separately from overall section color; document which regions were evaluated (general IHC practice; 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: exocrine glandular cells should stain (HPA: High in pancreatic exocrine glandular cells). HPA detects CHKB in all 45 scored tissues, so there is no HPA-supported negative tissue; use no-primary and isotype controls, and treat cells without signal on the positive slide as background comparators rather than validated CHKB-negative cells (HPA: no negative tissue rows).
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, a control immunoglobulin matched to the primary antibody’s host species and clonality, and CHKB-knockout material if available (standard IHC control practice). For pancreas chromogenic IHC, quench endogenous peroxidase and assess background from endogenous biotin if using biotin-based detection (standard IHC practice).
⚠️Feasibility: The selected A06297 caption shows paraffin-section IHC at 1:50 but does not report a fixative; a CHKB-specific fixation window and retrieval dependence are unreported in the supplied evidence (A06297 tissue-IHC caption). Compare antigen-retrieval conditions empirically; the supplied evidence does not establish that frozen sections or IF are easier (standard IHC practice; A06297 tissue-IHC caption). Pancreatic exocrine granules can complicate interpretation of background, so compare staining with the control slides (standard IHC practice).
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 by checking retrieval, cellular pattern, controls, and scoring before interpreting chromogenic signal.
How should I adjust retrieval when CHKB staining is weak in paraffin sections?
Start with citrate buffer at pH 6.0, heated to 95–98 °C for 20 min (page retrieval setting). Compare a known positive section across runs, using the same cooling interval and chromogenic detection conditions so retrieval is the main variable (general IHC practice). If staining remains weak, test a carefully controlled alternative retrieval buffer or heating interval on adjacent sections, while checking for tissue damage (general IHC practice). Score the expected cytoplasmic pattern rather than total brown signal: tissue IHC shows general cytoplasmic expression, and subcellular imaging supports cytosol localization (HPA tissue IHC; HPA subcellular).
Can fixation explain inconsistent CHKB staining between paraffin blocks?
The selected antibody image shows paraffin-embedded human colorectal carcinoma at 1:50, but its caption does not report the fixative (catalog antibody A06297 tissue-IHC caption). Target-specific sensitivity to fixative type or fixation duration is therefore unknown; neither the tissue staining profile nor CHKB topology establishes it (catalog antibody A06297 tissue-IHC caption; HPA tissue IHC; UniProt Q9Y259 topology). Compare blocks with documented processing histories, and hold retrieval, antibody incubation, and detection conditions constant during the comparison (general IHC practice). If a block stains poorly, inspect morphology and include a concurrently processed positive control before attributing the result to CHKB loss (general IHC practice).
Where should convincing CHKB staining appear within positive cells?
Expect predominantly cytoplasmic chromogenic staining: tissue IHC describes general cytoplasmic expression, and ICC/IF localization is approved for the cytosol (HPA tissue IHC; HPA subcellular). CHKB has no annotated transmembrane segment, but UniProt does not assign a subcellular location, so assess compartment claims against the direct imaging evidence (UniProt Q9Y259 topology; UniProt Q9Y259 subcellular record; HPA subcellular). Inspect cellular outlines and the counterstain at matched magnification to distinguish cytoplasmic signal from pigment or precipitate (general IHC practice). Predominantly nuclear or sharp membrane staining warrants comparison with a positive control and a detection-only control before being called CHKB (HPA subcellular; general IHC practice).
Can an unknown antibody epitope affect interpretation of CHKB isoforms?
CHKB has 2 annotated isoforms, and the supplied product caption does not identify the antibody epitope or establish isoform coverage (UniProt Q9Y259 isoforms; catalog antibody A06297 tissue-IHC caption). Report the result as antibody-detected CHKB staining unless epitope mapping or isoform-specific validation supports a narrower claim (general IHC practice). CHKB lacks an annotated transmembrane segment and glycosylation sites, while an N-acetylalanine is reported at residue 2; these annotations do not establish which epitope remains accessible after processing (UniProt Q9Y259 topology; UniProt Q9Y259 glycosylation and modified-residue records). For discordant specimens, compare independently validated epitopes on adjacent sections and document any difference in cytoplasmic pattern (general IHC practice; HPA tissue IHC).
How can IF help assess CHKB signal alongside a cell-type marker?
On the separate IF/ICC workflow, pair CHKB with a validated exocrine glandular cell marker when examining pancreas, where those cells show high tissue-IHC staining (HPA tissue IHC; general IF practice). Choose spectrally separated fluorophores and inspect an unstained section to place the CHKB channel away from strong tissue autofluorescence (general IF practice). CHKB is supported as cytosolic and has no annotated transmembrane segment, so permeabilize the plasma membrane sufficiently for intracellular antibody access while checking morphology (HPA subcellular; UniProt Q9Y259 topology; general IF practice). The epitope is unspecified in the supplied caption, so establish IF fixation and permeabilization conditions with IF-specific controls rather than transferring the paraffin-section image conditions (catalog antibody A06297 tissue-IHC caption; general IF practice).
What should I check when diffuse brown signal obscures CHKB staining?
First compare a detection-only control with the stained section to identify chromogen deposition from the detection system (general IHC practice). For peroxidase-based DAB detection, check the peroxidase block, then optimize protein blocking, antibody concentration, washes, and chromogen development while retaining the same positive control (general IHC practice). The selected catalog image used antibody at 1:50 in paraffin-embedded human colorectal carcinoma; that caption does not establish an optimal concentration for every specimen (catalog antibody A06297 tissue-IHC caption). Judge improvement by preserved cytoplasmic contrast within cells, consistent with the tissue staining profile, rather than by a lower whole-section brown intensity alone (HPA tissue IHC; general IHC practice).
How should I quantify CHKB IHC across sections with different cellularity? ⚠ ANSWER MARKED FOR VERIFICATION
Define the compartment and cell population before scoring; general cytoplasmic tissue staining and approved cytosolic localization support a cytoplasmic readout (HPA tissue IHC; HPA subcellular). Record the percentage of positive target cells and their staining intensity, then calculate an H-score as the sum of each intensity category multiplied by its percentage, using a prespecified scoring scale (general IHC practice). Normalize positive counts to the number of eligible cells, or stained area to analyzed viable tissue area in mm², while excluding folds and necrosis by a consistent rule (general IHC practice). Keep counterstain, imaging settings, threshold, and positive control consistent across batches (general IHC practice).
How can I distinguish true CHKB positivity from section artefacts?
A credible result places signal mainly in the cytoplasm of intact cells, matching the tissue-IHC profile and approved cytosolic localization (HPA tissue IHC; HPA subcellular). High staining in pancreatic exocrine glandular cells, placental decidual cells, or smooth muscle cells can inform positive-control selection, but it does not prove specificity in an individual section (HPA tissue IHC; general IHC practice). Treat predominantly nuclear or sharply membranous signal, staining confined to section edges or necrosis, and diffuse precipitate as findings requiring controls and morphology review (HPA subcellular; general IHC practice). For peroxidase-based detection, compare a detection-only control and confirm adequate peroxidase blocking before assigning brown signal to CHKB (general IHC practice).
Boster reagents
Best CHKB / Choline/ethanolamine kinase IHC Antibodies
The catalog shows CHKB IHC staining in a paraffin-embedded human colorectal carcinoma section; both antibodies list Human, Mouse and Rat reactivity, and one also lists IF (IHC image caption; catalog applications/reactivity).
Real IHC data
Anti-ChoKB (E324) CHKB Antibody
Cat # A06297
The rendered A06297 card shows IHC staining of paraffin-embedded human colorectal carcinoma tissue at 1:50 (A06297 IHC image caption). A06297-1 is listed for IHC and IF in Human, Mouse and Rat, but has no IHC or IF image and will not render as a card (A06297-1 catalog applications/reactivity/image captions; card list).
Which to pick: For tissue IHC, choose A06297: its own image documents paraffin-section staining, while the fixative is unreported (A06297 IHC image caption). For IF, consider A06297-1 because IF is listed; ICC performance and an IF figure are unreported (A06297-1 catalog applications/image captions). Either SKU lists Human, Mouse and Rat reactivity, but the only supplied tissue image is human (catalog reactivity; A06297 IHC image caption).
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 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 consistent across paraffin sections. (standard IHC practice; not target-specific); Caveat: Antibody staining and RNA show medium consistency (HPA tissue IHC); Isoform / epitope: 2 isoforms; epitope coverage is unspecified (UniProt); Regulation: Staining-linked regulation is not 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.