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CKB / Creatine kinase B-type · IHC design guide
Design Immunohistochemistry for CKB
Plan CKB chromogenic IHC on paraffin sections with the catalog antibody at 0.5–1 μg/ml (datasheet A01695-1). Compare staining in stomach parietal cells and CNS cells with cell-matched controls (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
CKB 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 in stomach and CNS; membrane in Purkinje cells (HPA tissue IHC) | |
| Staining pattern | Parietal and CNS cells: cytoplasm; Purkinje cells: membrane (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6 HIER, heat-mediated (datasheet A01695-1) | |
| Positive control | Cerebellum+4 more · see all | |
| Negative control | Adrenal gland+4 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A01695-1) | |
| Caveat | Staining varies with the cells sampled in each tissue (HPA tissue IHC) | |
| Regulation | Brain and intestine show tissue-enhanced RNA (HPA RNA) | |
| Isoform / epitope | No annotated isoforms or propeptide processing (UniProt) |
Section 1
Recommended CKB IHC & IF Protocols
The catalog antibody protocol uses citrate pH 6 heat retrieval (datasheet A01695-1). The four published CKB IHC protocols below provide additional conditions (PMC6221385; PMC10197964; PMC8494442; PMC7617069).
| Sample | Paraffin-embedded human colon cancer tissue; fixative not specified (datasheet A01695-1) |
| Fixation | Image fixative and duration unreported (datasheet A01695-1); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: Citrate pH 6, 20 min (datasheet A01695-1) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A01695-1) |
| Primary antibody | Rabbit anti-CKB, 0.5-1μg/ml (datasheet A01695-1) |
| Primary incubation | Overnight at 4 °C (datasheet A01695-1) |
| Detection | Streptavidin-biotin complex (SABC), DAB chromogen (datasheet A01695-1) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | CKB-positive staining in purkinje cells - cytoplasm/membrane of cerebellum (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression in parietal cells in stomach, and cells in the CNS. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6 heat retrieval (datasheet A01695-1). For FFPE optimization, compare the published 98°C for 25 minutes HIER condition (PMC7617069).
Section 2
What Is the Expected CKB Staining Pattern?
CKB staining should be predominantly cytoplasmic in stomach parietal cells and cells of the CNS, with strong signal reported in cerebellar Purkinje cells and cerebral cortical neuropil (HPA tissue IHC: Enhanced reliability; High in both sites). CKB is soluble, with no transmembrane segment, although UniProt also lists cell membrane and mitochondrial locations (UniProt P12277: localization and topology).
| Purkinje cell cytoplasm, sometimes with membrane-associated staining, is strong; cerebral cortical neuropil is also strong. | This matches the reported cell and compartment pattern (HPA tissue IHC: High in Purkinje cell cytoplasm/membrane and cerebral cortical neuropil). Compare the relevant structures, since neuropil signal cannot be scored as though it were neuronal cell-body staining. |
| Signal appears predominantly nuclear, while cytoplasm in an expected positive structure is weak or clear. | A nuclear-dominant pattern conflicts with supported cytosolic localization (HPA ICC-IF: Cytosol supported) and the reported tissue pattern (HPA tissue IHC: cytoplasmic expression). Check morphology and staining controls before interpreting it as CKB; membrane-associated Purkinje cell signal alone is compatible with HPA. |
| Unexpected cells stain strongly, such as hematopoietic cells in bone marrow or adrenal glandular cells. | Those cell populations were reported as not detected (HPA tissue IHC: bone marrow and adrenal gland). Cross-reactivity or endogenous chromogenic activity are possibilities (standard IHC practice). Verify cell identity and controls; an HPA negative observation is not proof that every specimen must remain negative. |
| Color covers the section diffusely, including spaces between cells and structures expected to have little signal. | Uniform haze obscures the cell-restricted pattern reported by HPA (HPA tissue IHC: cytoplasmic expression in selected cells). In chromogenic IHC, inadequate blocking, excess detection reagent, or residual endogenous enzyme activity can produce background (standard IHC practice); haze alone does not establish CKB expression. |
| No convincing staining appears in Purkinje cells or cerebral cortical neuropil, despite preserved tissue morphology. | These are useful positive comparators because both were scored High (HPA tissue IHC). A blank result warrants checking the run, antibody conditions, retrieval and detection (standard IHC practice). It does not establish biological absence of CKB until the positive control works. |
💡Expected CKB appearanceCall a result positive when expected CNS structures show strong, mainly cytoplasmic signal—Purkinje cells may include membrane staining—while nuclear-dominant or uniform section-wide color prompts control review (HPA tissue IHC: High CNS pattern; HPA ICC-IF: Cytosol supported; standard IHC practice).
| Cell and tissue selection | Purkinje cells and cerebral cortical neuropil are High; collecting ducts, prostate glandular cells and rectal enterocytes are Medium (HPA tissue IHC). Compare matching cell types within each tissue. |
| Compartment and topology | Cytosol is the supported ICC-IF location; UniProt also lists membrane and mitochondrion, with mitochondrial localization specified for thermogenic fat cells (HPA ICC-IF; UniProt P12277). Do not expect mitochondrial staining in every tissue. |
| Muscle context | Cardiomyocytes and skeletal myocytes have Low CKB staining (HPA tissue IHC). MM is the major creatine kinase form in skeletal muscle and myocardium (UniProt P12277); overall muscle kinase activity is not a CKB IHC score. |
| IHC evidence and antibody choice | HPA reports Enhanced IHC validation for HPA001254 and CAB047313 (HPA antibodies); tissue-level Enhanced reliability reflects consistency with RNA data (HPA tissue IHC). Neither statement guarantees every specimen or run. |
| IF/ICC Q&A: what localization is supported? | Cytosol is supported, with images listed for HEK293, U-251MG and U2OS (HPA ICC-IF). Use that as a localization cross-check; this IHC-P section provides no IF/ICC protocol. |
| Situation | Likely cause | Next action |
|---|---|---|
| A known-positive CNS section is blank. | The result conflicts with High staining in Purkinje cells and cerebral cortical neuropil (HPA tissue IHC); a failed IHC run is possible (standard IHC practice). | Confirm tissue morphology and a working positive control, then review retrieval, primary-antibody dilution, incubation and chromogenic detection (standard IHC practice). No CKB-specific retrieval condition is supplied. |
| Staining is mainly nuclear. | This differs from supported cytosolic localization and reported cytoplasmic tissue expression (HPA ICC-IF; HPA tissue IHC). | Check the counterstain and compare a positive tissue control with a no-primary control (standard IHC practice). Score only staining that fits the expected cell and compartment pattern. |
| A section has widespread brown haze. | Background may arise from incomplete blocking, excess reagent or endogenous peroxidase in chromogenic IHC (standard IHC practice). | Review blocking and washes, inspect the no-primary control, and adjust detection conditions if controls show background (standard IHC practice). Reassess signal in the expected cells. |
| Bone marrow hematopoietic cells or adrenal glandular cells appear strongly positive. | Both populations were not detected in the HPA tissue images (HPA tissue IHC); cross-reactivity or endogenous chromogenic activity may explain an unexpected pattern (standard IHC practice). | Confirm cell identity and examine no-primary and positive tissue controls (standard IHC practice). Treat a new biological interpretation as provisional until the pattern is independently supported. |
| Heart or skeletal muscle looks weaker than CNS tissue. | Low staining was reported in cardiomyocytes and skeletal myocytes, versus High in selected CNS structures (HPA tissue IHC); MM predominates in muscle (UniProt P12277). | Use the High CNS structures to assess assay performance and score the specified cell types separately (HPA tissue IHC). Do not use weak muscle staining alone to declare assay failure. |
| A membrane rim or punctate signal seems inconsistent with diffuse cytoplasm. | HPA includes membrane staining in Purkinje cells, while UniProt lists membrane and mitochondrial localization, the latter in thermogenic fat cells (HPA tissue IHC; UniProt P12277). | Identify the stained cell type and look for its expected cytoplasmic component (HPA tissue IHC). Interpret a new punctate pattern cautiously; these sources do not establish that appearance in every tissue. |
Experiment evidence
IHC & IF images showing CKB staining
A01695-1 · IHCIHC analysis of CKB using anti-CKB antibody (A01695-1). CKB was detected in paraffin-embedded section of human colon cancer tissue. Heat med
A01695-1 · IHCIHC analysis of CKB using anti-CKB antibody (A01695-1). CKB was detected in paraffin-embedded section of human colon cancer tissue. Heat med
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000166165-CKB/tissue/cerebral+cortex#imid_532306
HPA · StomachHuman Protein Atlas IHC, Stomach. Source: https://www.proteinatlas.org/ENSG00000166165-CKB/tissue/stomach+2#imid_532166
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000166165-CKB/tissue/colon#imid_12745657
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000166165-CKB/tissue/liver#imid_532225
HPA · ICC-IF HEK293Human Protein Atlas immunofluorescence, HEK293, antibody HPA001254; green = CKB, blue = nucleus, red = microtubules.
HPA · ICC-IF HEK293Human Protein Atlas immunofluorescence, HEK293, antibody HPA001254; green = CKB, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for CKB IHC & IF
🧪Run cerebellum first and require staining in Purkinje-cell cytoplasm and membrane (HPA: High in Purkinje cells). Use adrenal gland glandular cells as the negative tissue (HPA: Not detected); on the cerebellum slide, treat non-Purkinje cells with no signal above control background as internal negative references, without assuming every such cell lacks CKB.
Positive control tissue: Cerebellum (Purkinje cells - cytoplasm/membrane, HPA High)
Negative control tissue: Adrenal gland (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show CKB in HEK293, U-251MG, U2OS, with annotated localisation: Cytosol (supported) (HPA subcellular).
Technical controls: Include a no-primary (secondary-only) control, a concentration-matched rabbit IgG isotype control with matching clonality where known (selected-SKU caption: rabbit primary), and a CKB knockout sample or validated peptide-block control. For the cerebellum DAB assay, block endogenous peroxidase and assess endogenous biotin background because the caption uses biotin-based detection (selected-SKU caption: biotinylated secondary, SABC and DAB).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected-SKU paraffin-section caption does not state the fixative (selected-SKU caption: fixative unreported). The demonstrated IHC procedure uses heat retrieval in citrate buffer at pH 6 for 20 minutes (selected-SKU caption); the supplied evidence does not establish whether frozen sections or IF are easier. In cerebellum, score Purkinje-cell staining against surrounding neural tissue and the background controls (HPA: High in Purkinje-cell cytoplasm/membrane).
HPA tissue IHC evidence for CKB
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 |
|---|---|---|---|---|
| Cerebellum | Purkinje cells - cytoplasm/membrane | High | Protein (IHC) | HPA → |
| Cerebral cortex | Neuropil | High | Protein (IHC) | HPA → |
| Caudate | Glial cells | Medium | Protein (IHC) | HPA → |
| Hippocampus | Glial cells | Medium | Protein (IHC) | HPA → |
| Kidney | Collecting ducts | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adrenal gland | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Breast | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | Not detected | Protein (IHC) | HPA → |
| Cervix | Glandular cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced CKB IHC Tips
Troubleshoot CKB staining in paraffin-section chromogenic IHC using the catalog antibody’s documented conditions, compartment expectations, and matched controls.
What retrieval conditions should I start with for CKB in paraffin sections?
Start with heat-mediated citrate retrieval at pH 6 for 20 minutes on paraffin sections (datasheet A01695-1). Keep section thickness, heating, cooling, and detection consistent while assessing retrieval, since excessive heating can damage morphology (standard IHC practice). The documented example then used 10% goat serum, 1 µg/ml primary overnight at 4°C, and a biotinylated secondary with DAB detection (datasheet A01695-1). If staining remains weak, compare another retrieval buffer only as a fallback, using matched positive sections and a no-primary control to judge whether signal improves without added background (standard IHC practice).
Could fixation explain weak or inconsistent CKB staining?
The selected paraffin-section caption does not state a fixative, so CKB-specific fixation sensitivity is unknown (datasheet A01695-1). Record each block’s fixative and fixation interval, then compare matched sections under the documented citrate pH 6, 20-minute retrieval and 1 µg/ml primary conditions (datasheet A01695-1; standard IHC practice). If staining differs, inspect tissue preservation and run a concurrently processed positive control before assigning the difference to fixation (standard IHC practice). Paraffin embedding alone does not establish which fixative was used or how well this antibody tolerates it (datasheet A01695-1; standard IHC practice).
Which staining compartment should count as plausible CKB signal?
Expect mainly cytoplasmic signal: CKB is supported in the cytosol, and tissue IHC reports cytoplasmic expression in stomach parietal cells and CNS cells (HPA subcellular; HPA tissue IHC). Purkinje cells show high cytoplasmic or membrane staining, while cerebral cortex neuropil is also high (HPA tissue IHC). UniProt additionally reports cell-membrane localization and mitochondrial localization in thermogenic fat cells, so a compartment call should account for cell context rather than assume every punctum is mitochondrial (UniProt P12277 localization). Compare the observed pattern with a positive control and tissue morphology; isolated nuclear staining warrants scrutiny because the supplied localization evidence does not establish a nuclear pool (HPA subcellular; UniProt P12277 localization; standard IHC practice).
Can this stain distinguish CKB isoforms, complexes, or modified epitopes?
The record lists 0 CKB isoforms, so an isoform-specific interpretation is unsupported by these data (UniProt P12277 isoforms). CKB can form BB or MB dimers, but a tissue stain for the B chain alone does not identify which dimer produced a positive cell (UniProt P12277 subunit; standard IHC interpretation). The supplied caption gives no antibody epitope, so do not assign staining differences to a particular domain or modification (datasheet A01695-1). CKB has documented residues including phosphotyrosine 125 and phosphoserine 199; their presence in the record does not establish that either changes this antibody’s binding (UniProt P12277 modified residues; standard IHC interpretation).
How should I investigate a CKB pattern with multiplex IF?
Use IF as a separate comparison to the chromogenic IHC result, pairing CKB with a marker for the expected cell type, such as a Purkinje-cell marker when examining cerebellum (HPA tissue IHC; standard IF practice). Choose spectrally separated fluorophores and consider a red or far-red CKB channel where tissue autofluorescence complicates shorter-wavelength signal (standard IF practice). Because supported CKB signal is cytosolic and the protein has no transmembrane segment, assess permeabilisation sufficient to expose intracellular epitopes while preserving morphology (HPA subcellular; UniProt P12277 topology; standard IF practice). Include single-label and no-primary controls before treating apparent overlap as colocalization (standard IF practice).
How can I reduce diffuse or misleading chromogenic background?
Begin with the documented 10% goat-serum block and 1 µg/ml primary condition, then compare a no-primary section processed through the same detection steps (datasheet A01695-1; standard IHC practice). The selected image used a biotinylated secondary, streptavidin–biotin complex, and DAB, making endogenous biotin and endogenous peroxidase useful background checks for this workflow (datasheet A01695-1; standard IHC practice). Apply a peroxidase block and inspect whether background persists without primary antibody before changing the CKB antibody concentration (standard chromogenic IHC practice). When signal is diffuse, assess washing, blocking, and DAB development time against the matched control while retaining recognizable cell boundaries (standard IHC practice).
How should I quantify CKB staining across tissue sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring, because reported CKB patterns include Purkinje-cell cytoplasm or membrane, cortical neuropil, and collecting ducts (HPA tissue IHC). For discrete cells, record the percentage positive and staining intensity, or calculate an H-score from intensity classes 0–3 with a possible range of 0–300 (standard IHC practice). Normalize positive counts to the number of eligible cells, or report stained area per mm² of analyzable tissue when cell boundaries cannot be scored reliably (standard IHC practice). Hold retrieval, imaging, threshold, and region selection consistent, and exclude folds or necrosis before comparing sections (standard IHC practice).
How do I distinguish true CKB staining from artefact?
A credible result should fit tissue morphology and the predominantly cytosolic CKB pattern, with the documented cell context considered when membrane or neuropil signal appears (HPA subcellular; HPA tissue IHC). Compare a known positive region, such as Purkinje cells, with a no-primary control and the corresponding counterstained morphology (HPA tissue IHC; standard IHC practice). Edge-only staining, necrotic deposits, or signal appearing equally without primary antibody points toward processing or detection artefact rather than interpretable CKB distribution (standard IHC practice). Because the documented chromogenic method uses DAB after biotin-based detection, evaluate endogenous peroxidase and biotin contributions before calling unexpected brown staining positive (datasheet A01695-1; standard IHC practice).
Boster reagents
Best CKB / Creatine kinase B-type IHC Antibodies
A01695-1 has IHC images from paraffin sections of human colon cancer and mouse and rat kidney (catalog image captions). M01695 lists IF/ICC among its applications, but has no IF image (catalog applications; image inventory).
Real IHC data
Anti-Creatine kinase B type/CKB Antibody ®
Cat # A01695-1
A01695-1 lists IHC for human, mouse and rat and has paraffin-section images from human colon cancer and mouse and rat kidney (catalog applications, reactivity and image captions). M01695 lists IHC and IF/ICC for human, mouse and rat, but has no IHC or IF image in the payload (catalog applications, reactivity and image inventory).
Which to pick: For paraffin-section IHC, choose A01695-1: its own caption documents citrate retrieval at pH 6 for 20 minutes and 1 μg/ml primary antibody overnight at 4°C; the fixative is unreported (A01695-1 image caption). For IF/ICC, M01695 is the listed option, with a 1:50 IF dilution and monoclonal clone AFOC-3, but no IF image is supplied (M01695 catalog fields and image inventory). Both list human, mouse and rat reactivity; A01695-1 supplies IHC images across all three species, while M01695 supplies no IHC image (catalog reactivity and image inventory).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P12277 (KCRB_HUMAN, Creatine kinase B-type).
- Human Protein Atlas. CKB tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. CKB subcellular location (ICC-IF): Localized to the cytosol..
- Human Protein Atlas. CKB antibody validation summary (2 antibodies).
- Small Cell Carcinomas of the Uterine Cervix and Lung: Proteomics Reveals Similar Protein Expression Profiles. International journal of gynecological cancer : official journal of the International Gynecological Cancer Society 2018 — PMC6221385.
- Role of creatine shuttle in colorectal cancer cells. Oncotarget 2023 — PMC10197964.
- Therapeutic targeting of SLC6A8 creatine transporter suppresses colon cancer progression and modulates human creatine levels. Science advances 2021 — PMC8494442.
- The creatine-phosphagen system is mechanoresponsive in pancreatic adenocarcinoma and fuels invasion and metastasis. Nature metabolism 2020 — PMC7617069.
- PubMed PMID:3034271 — UniProt-cited evidence.
- PubMed PMID:3692484 — UniProt-cited evidence.
- PubMed PMID:2883200 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for CKB (Creatine kinase B-type). Expected localisation Cytoplasmic in stomach and CNS; membrane in Purkinje cells (HPA tissue IHC). Antigen retrieval: Citrate pH 6 HIER, heat-mediated (datasheet A01695-1). Validated IHC antibody: A01695-1 at 0.5-1μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Cerebellum (Purkinje cells - cytoplasm/membrane); negative Adrenal gland. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Paraffin-embedded human colon cancer tissue; fixative not specified (datasheet A01695-1); 2. Fixation: Image fixative and duration unreported (datasheet A01695-1); 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: Citrate pH 6 , 20 min (datasheet A01695-1); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A01695-1); 8. Primary antibody: Rabbit anti-CKB, 0.5-1μg/ml (datasheet A01695-1); 9. Primary incubation: Overnight at 4 °C (datasheet A01695-1). Watch for: Fixation: Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A01695-1); Caveat: Staining varies with the cells sampled in each tissue (HPA tissue IHC); Isoform / epitope: No annotated isoforms or propeptide processing (UniProt); Regulation: Brain and intestine show tissue-enhanced RNA (HPA RNA). Evidence: UniProt P12277, HPA tissue IHC · Enhanced, HPA ICC-IF · cytosol., Datasheet A01695-1, PMC6221385, PMC10197964. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/CKB/immunohistochemistry-ihc-guide.