CKB / Creatine kinase B-type · IHC design guide

Design Immunohistochemistry for CKB

Plan paraffin-section CKB IHC using the catalog antibody at 0.5–1 μg/mL (datasheet: A01695-1). Assess cytoplasmic staining in stomach parietal cells and CNS cells, with low staining expected in heart and skeletal myocytes (HPA tissue IHC).

Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use

Immunohistochemistry protocol sheet for CKB (IHC for CKB): expected localisation Cytoplasmic in stomach parietal and CNS cells (HPA tissue IHC), antibody A01695-1, validated IHC image, and IHC protocol steps — Printable **CKB** IHC protocol sheet — expected localisation Cytoplasmic in stomach parietal and CNS cells (HPA tissue IHC), antibody A01695-1, controls and protocol steps. Open the full CKB IHC guide →

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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 parietal and CNS cells (HPA tissue IHC)
	Staining pattern	Parietal-cell cytoplasm; Purkinje cytoplasm/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	Keep fixation consistent across sections. (standard IHC practice; not target-specific)
	Caveat	Heart and skeletal myocytes show low CKB staining (HPA tissue IHC)
	Regulation	Regulation not annotated (UniProt)
	Isoform / epitope	No annotated isoforms; mature chain spans residues 2–381 (UniProt)

Section 1

Recommended CKB IHC & IF Protocols

Compare the catalog antibody’s IHC-P protocol (datasheet A01695-1) with four published CKB IHC protocols (PMC6221385; PMC10197964; PMC8494442; PMC7617069).

Recommended immunohistochemistry (IHC-P) protocol parameters
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). A published alternative uses 98°C for 25 min in PT module 1 buffer (PMC7617069).

Section 2

What Is the Expected CKB Staining Pattern?

CKB is mainly cytosolic; UniProt also lists mitochondrial and cell membrane localization, with mitochondrial localization specified for thermogenic fat cells (UniProt P12277). In paraffin IHC, expect cytoplasmic staining in CNS cells and stomach parietal cells (HPA tissue IHC). HPA rates the tissue staining pattern Enhanced, citing consistency between antibody staining and RNA expression (HPA tissue IHC). CKB has no transmembrane segment (UniProt P12277 topology).

What am I looking at on my slide?
Strong Purkinje cell cytoplasmic/membrane staining or strong cerebral cortex neuropil staining (HPA tissue IHC).	These match HPA's High observations. Medium glial staining in caudate or hippocampus can also fit; compare the identified cell population, not just overall section darkness (HPA tissue IHC; general IHC practice).
Predominantly nuclear signal, with little cytoplasmic signal, in an expected positive cell population.	A nuclear-only pattern conflicts with the supported cytosolic location and reported tissue pattern (HPA subcellular ICC-IF; HPA tissue IHC). Check localization against the counterstain and staining controls before scoring it as CKB (general IHC practice).
Strong staining in adrenal gland glandular cells or bone marrow hematopoietic cells.	HPA reports CKB as Not detected in those specific cell populations (HPA tissue IHC). Unexpected signal warrants checks for antibody cross-reactivity and endogenous detection activity; a single discrepant section cannot establish which caused it (general IHC practice).
Diffuse chromogen across tissue and spaces, obscuring cell boundaries.	This is difficult to assign to the cytoplasmic cell patterns reported by HPA (HPA tissue IHC). Compare a no-primary control, blocking, washes and development time to assess nonspecific or detection background (general IHC practice).
No visible signal in cerebellar Purkinje cells or cerebral cortex neuropil.	Both are High HPA observations, so a blank expected-positive region calls for a run-level check (HPA tissue IHC). Confirm tissue identity and examine retrieval, antibody incubation and detection controls before interpreting other negatives (general IHC practice).

💡Expected CKB appearanceCall positive CKB when staining follows the identified cell's cytoplasmic pattern—strong in Purkinje cells or cortical neuropil (HPA tissue IHC); nuclear-only or diffuse, cell-independent color is suspect (HPA subcellular ICC-IF; general IHC practice).

How each factor affects the staining
Tissue and cell context	HPA reports Medium staining in kidney collecting ducts, prostate glandular cells, rectal enterocytes and stomach glandular cells, alongside High CNS observations (HPA tissue IHC). Score the named cells separately from surrounding tissue (general IHC practice).
Localization and topology	Cytosol is the supported HPA ICC-IF location; UniProt additionally lists mitochondrion and cell membrane and specifies mitochondrial localization in thermogenic fat cells (HPA subcellular ICC-IF; UniProt P12277). No transmembrane segment is annotated (UniProt P12277 topology).
Isoenzyme context	CKB forms dimers with B or M chains; UniProt describes MM as major in skeletal muscle and myocardium, with MB in myocardium (UniProt P12277). HPA reports Low CKB staining in cardiomyocytes and skeletal myocytes (HPA tissue IHC).
Antibody evidence	Target-specific fixation effects are not established by the supplied assay evidence. Verify with a matched IHC source before attributing a result to fixation.
What should IF/ICC show?	Predominantly cytosolic signal is the supported HPA ICC-IF localization; HPA001254 has Supported ICC status (HPA subcellular ICC-IF; HPA antibody validation). Treat organelle or membrane assignments as context dependent under the UniProt annotation (UniProt P12277).

Why is my staining missing, weak or wrong?
Situation	Likely cause	Next action
Expected-positive CNS region is blank.	A blank Purkinje cell or cortical neuropil region conflicts with HPA's High observations (HPA tissue IHC); a failed staining run is possible (general IHC practice).	Verify the region and a same-run positive control, then review retrieval, primary incubation and chromogenic detection steps (general IHC practice).
Only nuclei appear positive.	Nuclear-only staining does not match supported cytosolic localization (HPA subcellular ICC-IF).	Use the counterstain to confirm compartment boundaries; compare the no-primary control and reassess antibody concentration and detection background (general IHC practice).
Adrenal gland glandular cells stain strongly.	HPA records these cells as Not detected (HPA tissue IHC). Cross-reactivity or endogenous chromogenic activity may explain the discrepancy (general IHC practice).	Run a no-primary control and check the detection system's endogenous-activity block; repeat alongside an HPA High CNS region (general IHC practice; HPA tissue IHC).
Brown color is diffuse across the section.	Cell-independent color does not resemble HPA's localized cell observations (HPA tissue IHC); incomplete blocking, washing or excessive development may contribute (general IHC practice).	Compare no-primary and tissue controls, then review blocking, washes and chromogen development before scoring cells (general IHC practice).
Muscle is weaker than brain.	HPA reports Low staining in cardiomyocytes and skeletal myocytes, versus High staining in Purkinje cells and cortical neuropil (HPA tissue IHC).	Check the specified cell populations and same-run controls; do not use total creatine kinase activity to infer CKB staining, because B and M chains form different dimers (general IHC practice; UniProt P12277).
A negative cell population appears positive after longer development.	HPA's Not detected calls apply to specified cells, not every cell in an organ (HPA tissue IHC). Prolonged development can increase nonspecific color (general IHC practice).	Identify the stained cell type, compare no-primary and positive controls, and review development time before changing the interpretation (general IHC practice).

Source: UniProt P12277 Human Protein Atlas tissue Human Protein Atlas subcellular

Experiment evidence

IHC & IF images showing CKB staining

IHC analysis of CKB using anti-CKB antibody (A01695-1). CKB was detected in paraffin-embedded section of human colon cancer tissue. Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1μg/ml rabbit anti-CKB Antibody (A01695-1) overnight at 4°C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC)(Catalog # SA1022) with DAB as the chromogen.

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.

← 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: Purkinje cells should stain in the cytoplasm and at the membrane (HPA: High in Purkinje cells). Use adrenal gland as the negative tissue; its glandular cells are not detected (HPA: Not detected in adrenal gland glandular cells). On the cerebellum slide, assess cells outside the annotated Purkinje-cell staining pattern as internal background, without assuming every other cell type is CKB-negative (HPA: Purkinje cells - cytoplasm/membrane).

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 and a rabbit IgG isotype control matched to the primary antibody’s class and clonality; use a CKB knockout specimen as a biological negative where available (selected-SKU caption: rabbit primary and goat anti-rabbit secondary). Block or assess endogenous peroxidase and biotin in the cerebellum section because the reported IHC detection uses a streptavidin–biotin complex and DAB (selected-SKU caption: SABC/DAB).

⚠️Feasibility: A target-specific fixation window and fixation effect are unreported, and the selected-SKU paraffin-section caption does not state a fixative (selected-SKU caption: fixative unreported). The reported IHC procedure uses citrate retrieval at pH 6 for 20 minutes, but that colon-cancer example does not establish the optimal retrieval conditions for cerebellum (selected-SKU caption: colon-cancer paraffin section). ICC-IF shows supported cytosolic localization, but the supplied evidence does not establish whether frozen sections or IF are easier than paraffin IHC; evaluate neural-tissue background when scoring Purkinje-cell staining (HPA: Cytosol supported; HPA: High in cerebellar Purkinje cells).

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

Use the catalog antibody’s paraffin-section IHC evidence to troubleshoot CKB staining, and interpret the pattern against documented cell and compartment distributions (datasheet A01695-1; HPA tissue IHC; UniProt P12277).

What retrieval conditions should I try first when CKB staining is weak?

Start with heat-mediated retrieval in citrate buffer at pH 6 for 20 minutes (datasheet A01695-1). The catalog antibody detected CKB in a paraffin section of human colon cancer tissue after that treatment, with primary antibody at 1 μg/mL overnight at 4°C (datasheet A01695-1). If staining remains weak, check heating consistency and section adhesion before changing retrieval time, and compare any adjustment on matched sections (standard IHC practice). Record the actual heating and cooling conditions so a stronger signal can be weighed against tissue damage or increased background (standard IHC practice). The caption does not state a fixative, so it cannot establish fixation-specific retrieval performance (datasheet A01695-1).

Could fixation explain variable CKB staining between paraffin sections?

CKB-specific sensitivity to fixation is unknown from the supplied evidence; the catalog tissue caption identifies a paraffin section but does not report its fixative (datasheet A01695-1). Document each specimen’s fixative, fixation duration, processing history and section age before comparing staining intensity across cases (standard IHC practice). Run sections with different processing histories together using citrate retrieval at pH 6 for 20 minutes and the same detection conditions (datasheet A01695-1; standard IHC practice). If a signal differs, assess morphology and a concurrently stained reference section before attributing the change to fixation (standard IHC practice). Cytoplasmic HPA staining and CKB sequence features do not measure fixation sensitivity (HPA tissue IHC; UniProt P12277).

Which staining compartment is plausible for CKB in tissue IHC?

Expect predominantly cytoplasmic staining: CKB is annotated in the cytosol, with mitochondrial and cell-membrane localisation also reported (UniProt P12277). HPA describes cytoplasmic expression in stomach parietal cells and CNS cells, including high cytoplasm or membrane staining in cerebellar Purkinje cells (HPA tissue IHC). Assess membrane-associated signal alongside cytoplasmic signal because CKB has no transmembrane segment; a continuous membrane outline alone needs independent support (UniProt P12277; standard IHC practice). Mitochondrial localisation is specifically described for thermogenic fat cells and should not be assumed for every tissue (UniProt P12277). Compare each compartment with cell morphology and a negative control before calling an unexpected pattern positive (standard IHC practice).

How can epitope choice affect CKB specificity and staining?

The record lists 0 CKB isoforms, but CKB can form dimers containing B or M chains; the epitope recognised by this antibody is not supplied (UniProt P12277; datasheet A01695-1). Check the antibody’s documented immunogen or epitope before assuming that it distinguishes CKB from the muscle-type chain (standard IHC practice). CKB contains an N-terminal phosphagen kinase domain at residues 11–98 and a C-terminal domain at 125–367 (UniProt P12277). Reported modifications include phosphorylation at residues 4, 35, 125 and 199, plus nitration at 269; their effect on this antibody’s staining is unknown (UniProt P12277). Validate a questionable pattern with an independent CKB-specific reagent or orthogonal expression evidence (standard IHC practice).

How should I investigate a CKB pattern with multiplex IF?

Treat IF as a separate assay: HPA supports cytosolic localisation in ICC/IF images from HEK293, U-251MG and U2OS cells, while the catalog caption documents chromogenic tissue IHC (HPA subcellular; datasheet A01695-1). In tissue, pair CKB with a validated marker for the expected cell population, such as Purkinje cells when examining cerebellum (HPA tissue IHC; standard IF practice). Choose spectrally separated fluorophores, favouring a red or far-red CKB channel if shorter wavelengths show tissue autofluorescence, and inspect single-stain controls (standard IF practice). For an intracellular CKB epitope, optimise permeabilisation after fixation and compare signal with the no-primary control; the epitope’s accessibility is undocumented (UniProt P12277; standard IF practice). Do not transfer the IHC caption’s incubation settings directly to IF (datasheet A01695-1).

What should I check when CKB DAB staining is diffuse?

The catalog tissue example used 10% goat serum block, 1 μg/mL primary antibody overnight at 4°C, and a biotinylated secondary for 30 minutes at 37°C (datasheet A01695-1). When background is diffuse, inspect no-primary and secondary-only sections, then assess blocking, washing and primary concentration on matched sections (standard IHC practice). Its streptavidin–biotin DAB detection can require checks for endogenous biotin; peroxidase blocking is a general chromogenic workflow step, not CKB-specific evidence (datasheet A01695-1; standard IHC practice). Compare suspicious staining with the expected cytoplasmic distribution and tissue morphology before increasing retrieval or detection strength (UniProt P12277; standard IHC practice). Keep chromogen development consistent across comparison slides so background and signal remain interpretable (standard IHC practice).

How should I score CKB staining across tissue sections? ⚠ ANSWER MARKED FOR VERIFICATION

Define the cell population and compartment before scoring, because HPA reports high cytoplasm or membrane staining in Purkinje cells and no detection in colon endocrine cells (HPA tissue IHC). For cell-based comparisons, record percent positive cells and intensity grades, then calculate an H-score from 0–300 using the same thresholds across slides (standard IHC practice). For spatial comparisons, measure positive-cell density per mm² of viable tissue and report the analysed area (standard IHC practice). Normalise comparisons to the relevant cell count or viable tissue area, and exclude folds, edges and necrosis under predefined rules (standard IHC practice). Match retrieval, imaging and DAB development between batches, and retain representative controls alongside the numerical scores (standard IHC practice).

How can I distinguish true CKB staining from artefact?

Give greatest weight to staining in a plausible cell and compartment: HPA reports CNS and stomach cytoplasmic expression, while UniProt identifies cytosolic CKB with additional mitochondrial and membrane localisation (HPA tissue IHC; UniProt P12277). The catalog caption demonstrates DAB staining in a human colon cancer paraffin section, but does not establish that every cell in that section expresses CKB (datasheet A01695-1). A nuclear-only pattern, strong tissue-edge staining or signal confined to necrosis warrants control review before interpretation (UniProt P12277; standard IHC practice). Compare no-primary controls and peroxidase-blocked sections to identify nonspecific detection or endogenous enzyme activity (standard IHC practice). Check unexpected cell populations against morphology and independent expression evidence rather than assigning positivity from DAB colour alone (standard IHC practice).

Boster reagents

Best CKB / Creatine kinase B-type IHC Antibodies

The catalog covers human, mouse and rat CKB (catalog reactivity). IHC images show paraffin sections; no IF image is supplied (A01695-1 image captions; catalog IF images).

Real IHC data

Anti-Creatine kinase B type/CKB Antibody ®

Cat # A01695-1

View product →

A01695-1 has IHC images from human colon cancer and mouse and rat kidney paraffin sections (A01695-1 IHC image captions). M01695 lists IHC and IF/ICC for human, mouse and rat, but supplies no IHC or IF image (M01695 catalog applications, reactivity and image fields).

Which to pick: For tissue IHC, choose A01695-1: its own caption documents citrate pH 6 retrieval and staining of a human colon cancer paraffin section; the fixative is unreported (A01695-1 IHC caption). For IF/ICC, M01695 is the listed option, but its IF/ICC performance needs validation because no image is supplied (M01695 catalog applications and image fields). Both list human, mouse and rat reactivity; A01695-1 also has mouse and rat kidney IHC images, whereas M01695 has no IHC image (catalog reactivity; A01695-1 and M01695 image fields).

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.