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CMTM4 / CKLF-like MARVEL transmembrane domain-containing protein 4 · IHC design guide
Design Immunohistochemistry for CMTM4
Plan CMTM4 chromogenic IHC in paraffin sections using thyroid and parathyroid glandular cells as high-staining references (HPA tissue IHC). Assess cytoplasmic and membranous staining while accounting for presumed off-target binding reported in tissue IHC (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
CMTM4 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 and membranous staining (HPA tissue IHC) | |
| Staining pattern | Thyroid and parathyroid glandular cells: cytoplasmic and membranous (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen) | |
| Positive control | Parathyroid gland+4 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 paraffin sections. (standard IHC practice; not target-specific) | |
| Caveat | Presumed off-target binding was observed and disregarded (HPA tissue IHC) | |
| Regulation | Expression regulation is unreported (UniProt) | |
| Isoform / epitope | 3 isoforms; verify whether the epitope is extracellular or cytoplasmic (UniProt) |
Section 1
Recommended CMTM4 IHC & IF Protocols
Compare the catalog antibody's IHC-P protocol with 3 published CMTM4 IHC protocols (PMC10869054; PMC4609138; PMC7300202).
| Sample | Paraffin-embedded human breast carcinoma tissue; fixative not specified (datasheet A14124) |
| Fixation | Image fixative and duration unreported (datasheet A14124); 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-CMTM4, 1:100 (datasheet A14124) |
| 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 | CMTM4-positive staining in glandular cells of parathyroid gland (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic and membranous expression, most abundant in thryoid and parathyroid glands. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6.0 heat retrieval at 95–98 °C for 20 min (page antigen-retrieval setting).
Section 2
What Is the Expected CMTM4 Staining Pattern?
In paraffin-section IHC, expect cytoplasmic and membranous CMTM4 staining, strongest in thyroid and parathyroid glandular cells (HPA: tissue IHC). Its four transmembrane segments support a membrane-associated pattern (UniProt Q8IZR5: topology). HPA rates the tissue pattern Enhanced but reports presumed off-target binding that it disregarded (HPA: tissue IHC reliability); interpret unexpected staining with that caveat.
| Clear membranous and cytoplasmic staining in thyroid or parathyroid glandular cells (HPA: High in both). | This matches the reported positive pattern and provides a useful comparison for weaker samples (HPA: tissue IHC). Assess the glandular cells themselves; a dark area elsewhere does not establish the expected cell pattern (HPA: tissue IHC). |
| Predominantly nuclear staining, with little glandular-cell membrane or cytoplasmic signal. | Nuclear localisation is outside the reported tissue pattern (HPA: cytoplasmic and membranous) and the membrane annotation (UniProt Q8IZR5). Treat it as suspect staining and compare it with controls before scoring it as CMTM4 (general IHC practice). |
| Strong staining in adipocytes or oral-mucosa squamous epithelial cells (HPA: Not detected). | Those cell types are reported as not detected, so strong staining conflicts with the reference pattern (HPA: tissue IHC). Consider off-target antibody binding or endogenous detection activity; HPA specifically notes presumed off-target binding in its reliability assessment (HPA: tissue IHC; general IHC practice). |
| Diffuse chromogen over tissue and background, without a clear cell boundary or glandular-cell pattern. | A widespread haze cannot be read as the reported cytoplasmic and membranous distribution (HPA: tissue IHC). Check background and detection controls before assigning cellular localisation (general IHC practice). |
| No convincing glandular-cell signal in thyroid or parathyroid tissue (HPA: High in both). | This is discordant with the reported positive tissues (HPA: tissue IHC). First assess tissue preservation and whether the staining run worked using appropriate controls; a failed run cannot establish biological absence (general IHC practice). |
💡Expected CMTM4 appearanceCall a result positive when thyroid or parathyroid glandular cells show strong cytoplasmic and membranous staining (HPA: High; tissue IHC); isolated nuclear or strong not-detected-cell staining is suspect (HPA: tissue IHC; UniProt Q8IZR5: membrane).
| Compartment and topology | CMTM4 has four transmembrane segments and a MARVEL domain (UniProt Q8IZR5: topology, domain). HPA tissue IHC reports cytoplasmic and membranous staining; membrane association does not require every positive profile to appear exclusively at the cell surface (HPA: tissue IHC). |
| Choice of reference cells | Thyroid and parathyroid glandular cells are High; adrenal glandular cells and bronchial respiratory epithelial cells are Medium (HPA: tissue IHC). Compare like cell types when judging intensity, and avoid treating a whole section as uniformly positive (general IHC practice). |
| Evidence across assays | UniProt describes high expression in testis and prostate, while the supplied HPA tissue IHC highlights thyroid and parathyroid glands (UniProt Q8IZR5: tissue specificity; HPA: tissue IHC). These statements describe different evidence; neither supplies an IHC intensity for testis or prostate in this payload. |
| Antibody validation and isoforms | Two listed antibodies, HPA014704 and HPA023890, have Enhanced IHC status (HPA: antibody validation). CMTM4 has three isoforms, but the supplied record gives no antibody epitope or isoform coverage (UniProt Q8IZR5: isoforms; HPA: antibody validation); do not infer which isoform produces the stain. |
| IF/ICC Q: Where should signal appear? | A: HPA reports plasma membrane, Golgi apparatus and vesicles, each approved for ICC-IF (HPA: subcellular). This supports interpreting IF localisation, while the paraffin-section IHC reference remains cytoplasmic and membranous glandular-cell staining (HPA: tissue IHC). |
| Situation | Likely cause | Next action |
|---|---|---|
| Thyroid or parathyroid glandular cells show no signal (HPA: High in both). | The result conflicts with the reported IHC pattern; the supplied sources do not identify a CMTM4-specific fixation or retrieval failure (HPA: tissue IHC). | Verify section and run controls, then review the applied antigen-retrieval and detection conditions as general IHC steps; do not claim a CMTM4-specific retrieval requirement (general IHC practice). |
| Signal appears mainly in nuclei. | That compartment disagrees with HPA tissue IHC and the UniProt membrane annotation (HPA: tissue IHC; UniProt Q8IZR5: subcellular). | Check control sections and examine whether counterstain or background is being read as chromogen; score only interpretable cellular staining (general IHC practice). |
| Adipocytes or oral-mucosa squamous cells stain strongly (HPA: Not detected). | The pattern is unexpected for these cells; off-target binding or endogenous detection activity is possible (HPA: tissue IHC reliability; general IHC practice). | Compare an appropriate negative tissue and reagent control, and check the detection system's background controls before assigning the signal to CMTM4 (general IHC practice). |
| Brown haze obscures cell boundaries. | Nonspecific background can make localisation uninterpretable (general IHC practice). | Review blocking, washes and detection exposure against run controls; read glandular-cell membrane and cytoplasm only when boundaries remain clear (general IHC practice; HPA: tissue IHC pattern). |
| A medium-level tissue looks weaker than thyroid. | That difference can fit the reported levels: adrenal glandular cells and bronchial epithelium are Medium, thyroid glandular cells High (HPA: tissue IHC). | Compare the same cell type across sections and record intensity with its tissue context; do not use thyroid intensity as a universal threshold (HPA: tissue IHC; general IHC practice). |
| IF/ICC and paraffin IHC seem to show different detail. | ICC-IF resolves plasma membrane, Golgi and vesicles, while the supplied tissue IHC description groups staining as cytoplasmic and membranous (HPA: subcellular; tissue IHC). | Interpret each image against its own HPA assay annotation; use the tissue IHC pattern when scoring paraffin sections (HPA: tissue IHC; subcellular). |
Experiment evidence
IHC & IF images showing CMTM4 staining
A14124 · IHCImmunohistochemistryt analysis of paraffin-embedded human breast carcinoma, using CKLF4 Antibody. The lane on the right is blocked with the
A14124 · IFImmunofluorescence analysis of A549. 1, primary Antibody (red) was diluted at 1:200 (4°C overnight). 2, Goat Anti Rabbit IgG (H&L) - Alexa F
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000183723-CMTM4/tissue/cerebral+cortex#imid_3766712
HPA · StomachHuman Protein Atlas IHC, Stomach. Source: https://www.proteinatlas.org/ENSG00000183723-CMTM4/tissue/stomach+1#imid_3766620
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000183723-CMTM4/tissue/colon#imid_3766630
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000183723-CMTM4/tissue/liver#imid_3766639
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA014704; green = CMTM4, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA014704; green = CMTM4, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for CMTM4 IHC & IF
🧪Run parathyroid gland first and score its glandular cells for staining (HPA: parathyroid glandular cells High). Run adipose tissue as the negative and assess adipocytes for absent staining (HPA: adipocytes Not detected); on the parathyroid slide, assess morphologically identified non-glandular cells as candidate internal negatives, confirming that they lack specific staining rather than assuming they are negative (HPA: High assignment is for glandular cells).
Positive control tissue: Parathyroid gland (Glandular cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show CMTM4 in A-431, U-251MG, U2OS, with annotated localisation: Golgi apparatus (approved), Vesicles (approved), Plasma membrane (approved) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control and a host-species-matched isotype control for a monoclonal antibody, or matched nonimmune IgG for a polyclonal antibody (standard IHC practice). Include CMTM4 knockout material processed alongside the test sections if available, or test peptide competition with SKU A14124 (selected tissue-IHC caption: peptide block); check endogenous peroxidase background in the parathyroid section before interpreting chromogenic signal (standard IHC practice).
⚠️Feasibility: A target-specific fixation window and retrieval dependency are unreported in the supplied evidence; optimize antigen retrieval on serial paraffin sections (standard IHC practice). The selected SKU A14124 paraffin-section caption does not state a fixative, so its fixative is unreported (selected tissue-IHC caption). The evidence does not establish whether frozen sections or IF are easier for CMTM4, and it reports no parathyroid-specific artefact.
HPA tissue IHC evidence for CMTM4
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — 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 |
|---|---|---|---|---|
| Parathyroid gland | Glandular cells | High | Protein (IHC) | HPA → |
| Thyroid gland | Glandular cells | High | Protein (IHC) | HPA → |
| Adrenal gland | Glandular cells | Medium | Protein (IHC) | HPA → |
| Appendix | Glandular cells | Medium | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Oral mucosa | Squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
| Skin | Fibroblasts | Not detected | Protein (IHC) | HPA → |
| Soft tissue | Fibroblasts | Not detected | Protein (IHC) | HPA → |
| Vagina | Squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced CMTM4 IHC Tips
Troubleshoot CMTM4 staining in paraffin-section IHC by checking retrieval, compartment-specific signal, cell identity and controls before scoring.
Where should I start when CMTM4 staining is weak in paraffin sections?
Start with citrate pH 6.0 HIER at 95–98 °C for 20 min on paraffin sections (page retrieval setting). Check that sections remain attached and retrieval temperature is consistent across the batch before changing the antibody incubation (standard IHC practice). Compare a test section with a no-primary control and a parathyroid or thyroid glandular-cell control (HPA: High in both cell populations). Assess membranous and cytoplasmic staining together, since both are reported in tissue IHC (HPA: cytoplasmic and membranous expression). If signal remains weak, adjust one retrieval variable at a time while keeping detection and scoring conditions fixed (standard IHC practice).
Can fixation explain weak or uneven CMTM4 staining?
The supplied CMTM4 tissue caption identifies paraffin-embedded breast carcinoma but does not state the fixative (A14124 caption). Target-specific fixation sensitivity is therefore unknown; neither the tissue staining profile nor the protein topology establishes a preferred fixation condition (A14124 caption; HPA: tissue IHC; UniProt Q8IZR5 topology). Record the actual fixative and fixation duration for each specimen, then compare sections processed with the same retrieval and detection settings (standard IHC practice). If staining varies within a block, inspect preservation and section quality before changing antibody conditions (standard IHC practice). Keep the citrate pH 6.0, 20 min retrieval setting constant during that comparison (page retrieval setting).
Which staining compartments are plausible for CMTM4 in tissue IHC?
Score membranous and cytoplasmic signal separately, because both occur in the reported CMTM4 tissue pattern (HPA: cytoplasmic and membranous expression). Plasma membrane, Golgi apparatus and vesicles are supported subcellular locations, so a punctate intracellular component can be plausible (HPA: approved subcellular locations). CMTM4 has 4 transmembrane segments within a MARVEL domain spanning residues 49–176, consistent with membrane-associated staining (UniProt Q8IZR5 topology). Check whether staining follows intact cell boundaries or intracellular structures rather than section edges or damaged regions (standard IHC practice). Document the dominant compartment for each scored cell population instead of treating all brown signal as equivalent (standard IHC practice).
How should I troubleshoot an apparent epitope-dependent staining pattern?
CMTM4 has 3 recorded isoforms, but the supplied antibody caption does not map its epitope to any isoform (UniProt Q8IZR5 isoforms; A14124 caption). Its 4 transmembrane segments occupy residues 59–171, so epitope accessibility cannot be inferred from total protein expression alone (UniProt Q8IZR5 topology; standard IHC practice). Keep citrate pH 6.0 retrieval at 95–98 °C for 20 min as the initial comparison condition (page retrieval setting). If an epitope sequence becomes available, map it against each isoform before attributing discordant staining to splice variation (standard IHC practice). Report staining as antibody-detected CMTM4 rather than assigning an isoform without isoform-specific validation (standard IHC practice).
How can IF help resolve ambiguous chromogenic CMTM4 staining?
Use IF as a separate localisation check when chromogenic IHC cannot resolve membrane from intracellular staining (standard IHC/IF practice; HPA: cytoplasmic and membranous tissue expression). Multiplex CMTM4 with a validated marker for the cell population being assessed, such as glandular cells in a selected positive tissue (HPA: High in thyroid and parathyroid glandular cells). Choose fluorophores after examining an unstained section for tissue autofluorescence, and separate channels before interpreting colocalisation (standard IF practice). Match permeabilisation to the antibody epitope's membrane-facing side; that side is not specified for the catalog antibody here (A14124 caption; standard IF practice). Do not carry the paraffin tissue caption over as IF fixation or permeabilisation evidence (A14124 caption).
What should I check when CMTM4 DAB staining is diffuse?
Run a no-primary section to identify detection background, and inspect a matched section before DAB development for endogenous pigment (standard IHC practice). Include a peroxidase block before chromogenic detection and verify that its omission does not account for the observed signal (standard IHC practice). Review glandular-cell staining against a tissue reported as undetected in its relevant cell population, such as adipocytes in adipose tissue (HPA: Not detected in adipocytes). Diffuse staining across unrelated compartments warrants particular caution because presumed off-target binding was observed and disregarded in the tissue profile (HPA: Enhanced reliability description). Keep retrieval at citrate pH 6.0 for 20 min while troubleshooting background sources (page retrieval setting).
How should I quantify CMTM4 staining across tissue sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define viable cells of the relevant type before scoring, then record the percentage positive and intensity for membranous and cytoplasmic staining separately (standard IHC practice; HPA: cytoplasmic and membranous expression). An H-score can combine percentages at intensity levels 0–3; keep the threshold and scoring rules fixed across slides (standard IHC practice). For spatial comparisons, count positive cells per mm² of viable, annotated tissue and report the analysed area (standard IHC practice). Normalise cell-based results to the total eligible cells in the same compartment, excluding necrotic or damaged regions (standard IHC practice). Include a consistent positive control, such as thyroid glandular cells, when comparing staining batches (HPA: High in thyroid glandular cells).
How do I distinguish genuine CMTM4 signal from artefact?
A credible pattern places signal at cell membranes or in plausible intracellular structures within an identified cell population (HPA: tissue expression and approved subcellular locations). Compare the pattern with thyroid or parathyroid glandular cells reported as High and with cell populations reported as Not detected (HPA: tissue IHC). Treat signal restricted to section edges, necrotic areas or damaged cells as suspect, and inspect a no-primary section for detection artefact (standard IHC practice). Check endogenous enzyme activity with the peroxidase-blocked workflow before interpreting DAB deposits as antigen signal (standard IHC practice). Even plausible staining needs cautious attribution because the tissue profile notes presumed off-target binding (HPA: Enhanced reliability description).
Boster reagents
Best CMTM4 / CKLF-like MARVEL transmembrane domain-containing protein 4 IHC Antibodies
A14124 has IHC data from paraffin-embedded human breast carcinoma and IF data from A549 cells; the catalog lists Human and Mouse reactivity (IHC and IF image captions; catalog reactivity).
Real IHC data
Anti-CMTM4 Antibody
Cat # A14124
A14124 will render with an IHC image of paraffin-embedded human breast carcinoma (IHC image caption). Its application list includes IF, supported by an A549 fluorescence image, and its listed reactivity is Human and Mouse (catalog applications and reactivity; IF image caption).
Which to pick: For tissue IHC, choose A14124: its own image shows paraffin-embedded human breast carcinoma, but the fixative is unreported (IHC image caption). For IF/ICC, A14124 has listed IF use and an A549 fluorescence image; ICC is not separately listed (catalog applications; IF image caption). A14124 is polyclonal and lists Human and Mouse reactivity, although the supplied IHC and IF images do not demonstrate Mouse samples (catalog dilution text and reactivity; IHC and IF image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q8IZR5 (CKLF4_HUMAN, CKLF-like MARVEL transmembrane domain-containing protein 4).
- Human Protein Atlas. CMTM4 tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. CMTM4 subcellular location (ICC-IF): Localized to the plasma membrane, the Golgi apparatus and vesicles..
- Human Protein Atlas. CMTM4 antibody validation summary (2 antibodies).
- Prognostic significance of RKIP, TGM2, and CMTM4 expression in oral squamous cell carcinoma. Medicine 2024 — PMC10869054.
- CMTM4 inhibits gastric tumorigenesis and metastasis. Journal of gastrointestinal oncology 2024 — PMC11399846.
- CMTM4 is frequently downregulated and functions as a tumour suppressor in clear cell renal cell carcinoma. Journal of experimental & clinical cancer research : CR 2015 — PMC4609138.
- Expression Analysis of Canine CMTM6 and CMTM4 as Potential Regulators of the PD-L1 Protein in Canine Cancers. Frontiers in veterinary science 2020 — PMC7300202.
- PubMed PMID:12782130 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
- PubMed PMID:21269460 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for CMTM4 (CKLF-like MARVEL transmembrane domain-containing protein 4). Expected localisation Cytoplasmic and membranous staining (HPA tissue IHC). Antigen retrieval: Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen). Validated IHC antibody: A14124 at 1:100 (the product's own IHC validation image is shown). Controls (HPA): positive Parathyroid gland (Glandular cells); negative Adipose tissue. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Paraffin-embedded human breast carcinoma tissue; fixative not specified (datasheet A14124); 2. Fixation: Image fixative and duration unreported (datasheet A14124); 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-CMTM4, 1:100 (datasheet A14124); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across paraffin sections. (standard IHC practice; not target-specific); Caveat: Presumed off-target binding was observed and disregarded (HPA tissue IHC); Isoform / epitope: 3 isoforms; verify whether the epitope is extracellular or cytoplasmic (UniProt); Regulation: Expression regulation is unreported (UniProt). Evidence: UniProt Q8IZR5, HPA tissue IHC · Enhanced, HPA ICC-IF · plasma membrane, the Golgi apparatus and vesicles., Datasheet A14124, PMC10869054, PMC4609138. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/CMTM4/immunohistochemistry-ihc-guide.