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CKMT2 / Creatine kinase S-type, mitochondrial · IHC design guide
Design Immunohistochemistry for CKMT2
Plan CKMT2 paraffin-section IHC around cytoplasmic staining in heart and skeletal muscle (HPA tissue IHC). This guide covers fixation consistency, antibody dilution, controls and interpretation of possible off-target staining (HPA tissue IHC; datasheet: 1:50–1:200).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
CKMT2 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 staining (HPA tissue IHC); inner mitochondrial membrane target (UniProt) | |
| Staining pattern | High cardiomyocyte and medium skeletal myocyte cytoplasmic staining (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen) | |
| Positive control | Heart muscle+2 more · see all | |
| Negative control | Adipose tissue+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 | Presumed off-target binding complicates interpretation (HPA tissue IHC) | |
| Regulation | Sarcomere-specific expression (UniProt) | |
| Isoform / epitope | No listed isoforms; mature chain spans aa 40–419 (UniProt) |
Section 1
Recommended CKMT2 IHC & IF Protocols
The catalog antibody’s IHC-P protocol is accompanied by two published CKMT2 staining methods for paraffin sections (PMC10764887; PMC13006575).
| Sample | Paraffin-embedded human tonsil cancer tissue; fixative not specified (datasheet A11457-1) |
| Fixation | Image fixative and duration unreported (datasheet A11457-1); 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-CKMT2, 1:50-1:200 (datasheet A11457-1) |
| 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 | CKMT2-positive staining in cardiomyocytes of heart muscle (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression mainly in heart and skeletal muscle. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6.0 retrieval (page retrieval rule; PMC13006575). If staining needs optimization, evaluate heat-mediated EDTA pH 9.0 (PMC10764887).
Section 2
What Is the Expected CKMT2 Staining Pattern?
CKMT2 localises to the mitochondrial inner membrane without a transmembrane segment (UniProt P17540 topology). In paraffin section IHC, expect cytoplasmic staining in cardiomyocytes and skeletal myocytes (HPA tissue IHC). HPA reports high staining in cardiomyocytes and medium staining in skeletal myocytes, with Enhanced IHC reliability; its summary also notes presumed off target binding (HPA tissue IHC; HPA antibodies).
| Cardiomyocytes show strong cytoplasmic staining; skeletal myocytes show moderate cytoplasmic staining. | This matches the reported cell types, compartments and relative levels (HPA tissue IHC). Chromogenic IHC shows a cytoplasmic pattern; it does not resolve the mitochondrial inner membrane identified by UniProt (UniProt P17540 topology; general IHC practice). |
| Nuclei stain prominently while the expected cytoplasmic pattern is absent. | Nuclear localisation conflicts with the mitochondrial inner membrane assignment (UniProt P17540 topology). Review the counterstain, detection controls and antibody specificity before scoring nuclear colour as CKMT2 (general IHC practice). |
| Unexpected cell types stain, including distal tubules in kidney. | HPA reports high distal tubular staining, although UniProt describes CKMT2 as found only in heart and skeletal muscle (HPA tissue IHC; UniProt P17540 tissue specificity). Given HPA's off target caveat, treat this as unresolved staining, not confirmed CKMT2 expression (HPA tissue IHC). |
| Colour spreads across tissue structures or appears in negative controls. | A poorly confined signal cannot establish the reported cytoplasmic pattern (HPA tissue IHC; general IHC practice). Background may reflect nonspecific binding or endogenous detection activity; compare controls and examine where the colour is deposited (general IHC practice). |
| Cardiomyocytes show no signal in an otherwise interpretable section. | This differs from HPA's high cardiomyocyte staining (HPA tissue IHC). Check section quality, antibody and detection controls, and the laboratory's retrieval conditions before interpreting the result as absent CKMT2 (general IHC practice). |
💡Expected CKMT2 appearanceA positive result is strong cytoplasmic staining in cardiomyocytes or moderate cytoplasmic staining in skeletal myocytes (HPA tissue IHC); isolated nuclear or broad background colour is suspect because CKMT2 is assigned to the mitochondrial inner membrane (UniProt P17540 topology; general IHC practice).
| Compartment and resolution | CKMT2 is assigned to the mitochondrial inner membrane and has no transmembrane segment (UniProt P17540 topology). Interpret chromogenic staining at the cytoplasmic level; this method does not by itself verify inner membrane localisation (general IHC practice). |
| Tissue and antibody evidence | HPA reports Enhanced IHC for HPA051880 and medium consistency between staining and RNA data, while noting presumed off target binding (HPA antibodies; HPA tissue IHC). These qualifications matter when judging unexpected staining. |
| Processing and isoforms | UniProt lists a mature chain spanning residues 40–419 and no isoforms (UniProt P17540 processing; UniProt P17540 isoforms). The supplied record gives no antibody epitope, so it cannot establish whether processing changes antibody recognition. |
| Retrieval and detection | Antigen retrieval and checks for endogenous chromogenic activity are general paraffin IHC considerations (general IHC practice). The supplied HPA and UniProt records give no CKMT2 specific retrieval condition or fixation sensitivity. |
| IF/ICC Q&A: can its localisation be scored here? | HPA supplies no main ICC/IF location or cell line images for CKMT2 (HPA subcellular ICC-IF). UniProt supports a mitochondrial expectation, but this record provides no observed ICC/IF pattern to score against (UniProt P17540 topology; HPA subcellular ICC-IF). |
| Situation | Likely cause | Next action |
|---|---|---|
| Heart muscle is negative. | The result conflicts with high cardiomyocyte staining in HPA (HPA tissue IHC); the failed step is undetermined. | Confirm tissue identity and section integrity, then review retrieval, antibody application, detection and a working positive control (general IHC practice). |
| Skeletal muscle is weaker than heart muscle. | HPA grades skeletal myocytes medium and cardiomyocytes high (HPA tissue IHC). | Judge each tissue against its reported level and cytoplasmic distribution before calling skeletal muscle negative (HPA tissue IHC; general IHC practice). |
| Kidney distal tubules stain strongly. | HPA reports this staining despite UniProt's heart and skeletal muscle tissue specificity; HPA also notes presumed off target binding (HPA tissue IHC; UniProt P17540 tissue specificity). | Record the discrepancy, review controls and seek independent specificity evidence before attributing tubular colour to CKMT2 (general IHC practice). |
| Nuclear staining dominates. | This conflicts with the mitochondrial inner membrane assignment (UniProt P17540 topology); the source of the nuclear colour is undetermined. | Inspect a negative detection control, counterstain and antibody specificity, then score only interpretable compartmental staining (general IHC practice). |
| Diffuse staining obscures cell boundaries. | Background or endogenous detection activity can make cytoplasmic staining hard to distinguish (general IHC practice). | Compare negative controls and review blocking, antibody concentration, washing and detection conditions (general IHC practice). |
| A presumed negative tissue stains. | HPA lists adipocytes in adipose tissue as not detected, but its tissue summary notes presumed off target binding (HPA tissue IHC). A single unexpected positive does not identify its cause. | Check the matching cell type, negative controls and signal distribution; seek independent specificity evidence before revising the expected pattern (HPA tissue IHC; general IHC practice). |
Experiment evidence
IHC & IF images showing CKMT2 staining
A11457-1 · IHCImmunohistochemistry (IHC) analyzes of CKMT2/sMtCK (W262) pAb in paraffin-embedded human tonsil cancer tissue at 1:50.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000131730-CKMT2/tissue/cerebral+cortex#imid_14748500
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000131730-CKMT2/tissue/colon#imid_14772542
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000131730-CKMT2/tissue/liver#imid_14772370
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000131730-CKMT2/tissue/kidney#imid_14772215
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for CKMT2 IHC & IF
🧪Run heart muscle first: cardiomyocytes should stain (HPA: High in cardiomyocytes). Run adipose tissue as a negative, where adipocytes are not detected (HPA: Not detected in adipocytes); on the heart slide, use noncardiomyocyte cells as candidate internal negatives and look for background staining rather than cardiomyocyte-like granular cytoplasmic signal (HPA: High in cardiomyocytes; UniProt P17540: mitochondrial inner membrane).
Positive control tissue: Heart muscle (Cardiomyocytes, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA carries no ICC-IF cell line for CKMT2; derive a cell-line control from the positive tissue's cell type (Cardiomyocytes) and confirm it by RNA or western blot first.
Technical controls: Include a no-primary (secondary-only) control, an irrelevant immunoglobulin control matched to the primary antibody’s host species, class, and monoclonal or polyclonal format, and CKMT2 knockout material or cognate-peptide blocking to check specificity (standard IHC practice). For chromogenic heart sections, quench endogenous peroxidase and check endogenous biotin if using biotin-based detection (standard IHC practice).
⚠️Feasibility: The selected A11457-1 paraffin-section caption reports 1:50 but does not report its fixative; no target-specific fixation window or antigen-retrieval requirement is supplied (selected tissue-IHC caption). The supplied evidence does not establish whether frozen sections or IF/ICC are easier, and HPA lists no CKMT2 ICC-IF images (HPA subcellular: no ICC-IF image cell lines). Cardiac tissue autofluorescence can complicate IF interpretation (standard IF practice).
HPA tissue IHC evidence for CKMT2
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — Medium consistency between antibody staining and RNA expression data. Presumed off target binding observed and disregarded.). 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 |
|---|---|---|---|---|
| Heart muscle | Cardiomyocytes | High | Protein (IHC) | HPA → |
| Kidney | Distal tubules | High | Protein (IHC) | HPA → |
| Skeletal muscle | Myocytes | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Appendix | 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 → |
Section 3
Advanced CKMT2 IHC Tips
Troubleshoot CKMT2 staining in paraffin sections by checking retrieval, compartment, tissue context and controls before assigning a chromogenic signal to the target.
How should I retrieve CKMT2 in paraffin sections when staining is weak?
Start with citrate buffer at pH 6.0 for heat-induced retrieval at 95–98 °C for 20 minutes (page retrieval rule). Keep section thickness, cooling time and detection conditions consistent across the first comparison, then check a heart-muscle control for cardiomyocyte staining (HPA: High in cardiomyocytes). If staining remains weak, test a second retrieval condition on matched sections while retaining the citrate condition as the reference; altered retrieval can also raise background (standard IHC practice). Score the resulting cytoplasmic pattern against morphology, because CKMT2 is associated with the mitochondrial inner membrane and has no transmembrane segment (UniProt P17540 topology; HPA: cytoplasmic expression).
Could fixation explain absent CKMT2 staining?
CKMT2-specific sensitivity to fixative type or fixation duration is unknown from the supplied evidence; the paraffin-section caption does not state a fixative (A11457-1 caption). Record each block’s documented fixative, fixation duration and processing history, then compare sections processed alike before changing antibody or detection conditions (standard IHC practice). Include a heart-muscle section with cardiomyocytes as a positive comparator and run a no-primary control alongside the questioned section (HPA: High in cardiomyocytes; standard IHC practice). If only one block fails, investigate its processing and tissue preservation, but do not assign the failure to a CKMT2-specific fixation effect without a controlled comparison.
Where should convincing CKMT2 staining appear within a positive cell?
Expect a cytoplasmic staining pattern in relevant cells, consistent with the reported tissue-IHC profile and CKMT2’s mitochondrial inner-membrane location (HPA: cytoplasmic expression; UniProt P17540 location). A granular or organelle-rich cytoplasmic pattern can support this assignment, but chromogenic IHC alone does not resolve the two sides of that membrane (standard IHC interpretation). Review cardiomyocytes in heart muscle and myocytes in skeletal muscle as reference cell populations (HPA: High in cardiomyocytes; Medium in myocytes). Treat isolated nuclear staining or staining confined to tissue folds as suspect, and compare it with morphology and a no-primary control before scoring it (standard IHC practice).
How do processing and epitope placement affect CKMT2 IHC interpretation?
CKMT2 has a 419-amino-acid precursor and a reported mature chain spanning residues 40–419; no alternative isoforms are listed (UniProt P17540 processing; isoforms). Check the antibody’s documented immunogen or epitope before interpreting a negative section, since an epitope outside the mature chain could complicate detection (UniProt P17540 processing; standard IHC practice). The protein has no transmembrane segment and carries reported phosphorylation at residues 255 and 356 (UniProt P17540 topology; modified residues). Do not attribute a changed chromogenic pattern to either modification without epitope mapping and a controlled comparison, because the supplied IHC evidence does not establish modification-dependent staining.
How can IF help check a disputed CKMT2 IHC pattern?
On a separately validated IF section, multiplex CKMT2 with a marker that identifies the expected cell population, such as cardiomyocytes in heart muscle, then compare cell identity with the chromogenic result (HPA: High in cardiomyocytes; standard IF practice). Choose fluorophores after inspecting unstained tissue autofluorescence, and use separate single-stain controls to assess channel bleed-through (standard IF practice). Select permeabilisation only after establishing whether the antibody epitope is accessible from the cytoplasmic side or requires access across mitochondrial membranes (UniProt P17540: inner-membrane location; standard IF practice). An IF pattern can clarify cellular distribution, but this payload supplies no CKMT2 IF/ICC validation or epitope-side assignment (HPA subcellular: no ICC/IF images supplied).
What should I change when CKMT2 chromogenic staining is diffuse?
First compare the diffuse signal with a no-primary control and a documented positive section, keeping exposure to chromogen and counterstain consistent (standard IHC practice). Titrate the primary antibody and review blocking, washes and detection chemistry on matched sections; 1:50 is reported for the selected paraffin-section image, not established as a universal optimum (A11457-1 caption; standard IHC practice). Include a peroxidase-block step before peroxidase-based detection and inspect tissue without primary antibody for residual endogenous signal (standard chromogenic IHC practice). Be cautious with diffuse staining outside heart and skeletal muscle, while recognizing that HPA also reports high staining in kidney distal tubules (UniProt P17540 tissue specificity; HPA: High in distal tubules).
How should I score CKMT2 across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the scored cell population and compartment before reading slides: cytoplasm in cardiomyocytes or myocytes is supported by the tissue profile (HPA: cytoplasmic expression; High in cardiomyocytes; Medium in myocytes). For a cell-based comparison, record percent positive cells and intensity categories, then calculate an H-score from those categories using the same thresholds across sections (standard IHC scoring practice). Normalize counts to evaluable cells of the chosen type, or normalize positive area to viable tissue area in mm² when area is the prespecified endpoint (standard IHC quantification practice). Exclude folds, edges and necrotic regions by a documented rule, and report positive-control performance with each batch (standard IHC practice).
How can I distinguish true CKMT2 signal from an artefact?
Prioritize cytoplasmic staining in morphologically intact cardiomyocytes or skeletal myocytes, consistent with CKMT2’s mitochondrial location and the reported tissue pattern (UniProt P17540 location; HPA: cytoplasmic expression; High in cardiomyocytes; Medium in myocytes). Check an adjacent no-primary section when signal appears nuclear, concentrated at section edges or restricted to necrotic tissue; these patterns warrant artefact review (standard IHC practice). For peroxidase-based detection, examine the no-primary control after the peroxidase block to identify residual endogenous enzyme signal (standard chromogenic IHC practice). Interpret staining outside the expected cells cautiously: HPA reports high kidney distal-tubule staining but also notes presumed off-target binding in its tissue assessment (HPA: High in distal tubules; reliability description).
Boster reagents
Best CKMT2 / Creatine kinase S-type, mitochondrial IHC Antibodies
Anti-CKMT2 A11457-1 has a paraffin-section IHC image from human tonsil cancer tissue; IF is listed without an image (catalog image caption; catalog applications). Human, mouse and rat reactivity is listed (catalog reactivity).
Real IHC data
Anti-CKMT2 (W262) Antibody
Cat # A11457-1
A11457-1 has an IHC image of paraffin-embedded human tonsil cancer tissue at 1:50 (catalog image caption). A11457-1 lists IHC and IF applications and human, mouse and rat reactivity; no IF image is supplied (catalog applications; catalog reactivity; catalog IF images).
Which to pick: Choose A11457-1 for paraffin-section tissue IHC because its own image shows that preparation at 1:50; the fixative is unreported (catalog image caption). For IF/ICC, A11457-1 lists IF at 1:50–1:200, but supplies no IF image or ICC validation (catalog applications; catalog IF dilution; catalog IF images). It is the listed cross-species option for human, mouse and rat and is polyclonal (catalog reactivity; catalog dilution record).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P17540 (KCRS_HUMAN, Creatine kinase S-type, mitochondrial).
- Human Protein Atlas. CKMT2 tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. CKMT2 subcellular location (ICC-IF): Highest expression in SK-MEL-30: 2.9 nTPM.
- Human Protein Atlas. CKMT2 antibody validation summary (1 antibodies).
- Creatine kinase mitochondrial 2 promotes the growth and progression of colorectal cancer via enhancing Warburg effect through lactate dehydrogenase B. PeerJ 2024 — PMC11216189.
- Heterogeneity-based stratification identifies CKMT2 as a prognostic marker in osteosarcoma. Frontiers in cell and developmental biology 2026 — PMC13247562.
- Prognostic value of mitochondrial CKMT2 in Pan-cancer and its tumor immune correlation analysis. Scientific reports 2024 — PMC10764887.
- Metabolic profiling of the TME uncovers the contrasting impacts of CKMT2 and PDE2A in CRC progression and therapeutic response. Frontiers in pharmacology 2026 — PMC13006575.
- PubMed PMID:2324105 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
- PubMed PMID:2914937 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for CKMT2 (Creatine kinase S-type, mitochondrial). Expected localisation Cytoplasmic staining (HPA tissue IHC); inner mitochondrial membrane target (UniProt). Antigen retrieval: Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen). Validated IHC antibody: A11457-1 at 1:50-1:200 (the product's own IHC validation image is shown). Controls (HPA): positive Heart muscle (Cardiomyocytes); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human tonsil cancer tissue; fixative not specified (datasheet A11457-1); 2. Fixation: Image fixative and duration unreported (datasheet A11457-1); 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-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-CKMT2, 1:50-1:200 (datasheet A11457-1); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific); Caveat: Presumed off-target binding complicates interpretation (HPA tissue IHC); Isoform / epitope: No listed isoforms; mature chain spans aa 40–419 (UniProt); Regulation: Sarcomere-specific expression (UniProt). Evidence: UniProt P17540, HPA tissue IHC · Enhanced, HPA cell-line RNA · Highest expression in SK-MEL-30: 2.9 nTPM, Datasheet A11457-1, PMC10764887, PMC13006575. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/CKMT2/immunohistochemistry-ihc-guide.