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METTL14 / N(6)-adenosine-methyltransferase non-catalytic subunit METTL14 · IHC design guide
Design Immunohistochemistry for METTL14
Plan chromogenic METTL14 IHC in paraffin sections using nuclear staining as the readout (HPA tissue IHC). Lung alveolar type I cells and testis pachytene spermatocytes show high staining, while liver cholangiocytes show none detected (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
METTL14 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 | Nuclear staining in tissue (HPA tissue IHC) | |
| Staining pattern | Widespread nuclear staining across cell types (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A07389-2) | |
| Positive control | Lung+4 more · see all | |
| Negative control | Liver |
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 A07389-2) | |
| Caveat | Liver cholangiocytes may show no detectable staining (HPA tissue IHC) | |
| Regulation | Low tissue specificity; no regulator reported (HPA tissue IHC) | |
| Isoform / epitope | 0 annotated isoforms; one chain, residues 1–456 (UniProt) |
Section 1
Recommended METTL14 IHC & IF Protocols
The catalog antibody’s IHC-P protocol (datasheet A07389-2) is followed by published METTL14 staining protocols for breast and nucleus pulposus tissues (PMC12885814; PMC13197395).
| Sample | Paraffin-embedded human breast cancer tissue; fixative not specified (datasheet A07389-2) |
| Fixation | Image fixative and duration unreported (datasheet A07389-2); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: EDTA pH 8.0 (datasheet A07389-2); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A07389-2) |
| Primary antibody | Rabbit anti-METTL14, 2-5μg/ml (datasheet A07389-2) |
| Primary incubation | Overnight at 4 °C (datasheet A07389-2) |
| Detection | Streptavidin-biotin complex (SABC), DAB chromogen (datasheet A07389-2) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | METTL14-positive staining in alveolar cells type I of lung (HPA tissue IHC: High). HPA tissue profile: Ubiquitous nuclear expression. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA pH 8.0 for the catalog antibody (datasheet A07389-2). The published protocols use EDTA pH 9.0 or citrate pH 6.0 (PMC12885814; PMC13197395).
Section 2
What Is the Expected METTL14 Staining Pattern?
METTL14 is a nuclear protein with no transmembrane segment (UniProt Q9HCE5). In paraffin-section IHC, expect nuclear staining across many cell types: HPA describes ubiquitous nuclear expression and low tissue specificity (HPA tissue IHC). Alveolar type I cells and pachytene spermatocytes are reported as high-staining examples (HPA tissue IHC). HPA rates the tissue IHC pattern Supported, with medium consistency between antibody staining and RNA data (HPA tissue IHC).
| Distinct nuclear staining in the expected cells. | This matches the reported nuclear localisation (UniProt Q9HCE5; HPA tissue IHC). Judge intensity against the cell type: HPA reports high staining in alveolar type I cells and pachytene spermatocytes, but medium staining in several other listed cell types (HPA tissue IHC). A positive call should rest on identifiable nuclei, not colour elsewhere on the section (general IHC practice). |
| Predominantly cytoplasmic, membranous, or extracellular colour. | This conflicts with METTL14's nuclear localisation and lack of a transmembrane segment (UniProt Q9HCE5). Treat a compartment-only signal as suspect and inspect the negative control, counterstain, and staining distribution for background or nonspecific detection (general IHC practice). HPA's tissue IHC summary supports a nuclear pattern; it does not validate those alternative compartments (HPA tissue IHC). |
| Strong staining in a cell population expected to be weak or unstained. | Compare the same identified cell type, rather than the tissue as a whole (general IHC practice). HPA reports liver cholangiocytes as not detected and several other listed cell populations as low (HPA tissue IHC). Unexpected strong colour, especially outside nuclei, warrants a cross-reactivity or endogenous detection-activity check (general IHC practice). One discrepant field alone does not establish either cause. |
| Uniform haze or precipitate obscures nuclear boundaries. | A diffuse deposit cannot establish the reported nuclear pattern (UniProt Q9HCE5; HPA tissue IHC). Check whether colour also appears in a negative control, follows tissue edges, or covers cell-free areas; these observations help distinguish background from interpretable cellular staining (general IHC practice). Score only cells whose nuclei can be separated from surrounding colour (general IHC practice). |
| No detectable nuclear signal in a high-staining reference cell population. | Alveolar type I cells and pachytene spermatocytes are high-staining examples in the HPA tissue IHC record (HPA tissue IHC). First confirm that the relevant cells are present and recognisable, then assess the run controls and detection workflow (general IHC practice). An absent signal may reflect an assay failure, but the supplied sources do not identify a METTL14-specific fixation or retrieval cause. |
💡Expected METTL14 appearanceCall a result positive when identifiable nuclei stain in the sampled cells, with high signal possible in alveolar type I cells or pachytene spermatocytes (UniProt Q9HCE5; HPA tissue IHC); isolated cytoplasmic colour or diffuse haze is suspect (general IHC practice).
| Compartment and target form | METTL14 is annotated as nuclear and has no transmembrane segment or signal peptide (UniProt Q9HCE5). These annotations support a nuclear interpretation; they provide no basis for expecting a surface or secreted staining pattern. |
| Variation among cell populations | HPA describes ubiquitous nuclear expression but reports levels from high in alveolar type I cells and pachytene spermatocytes to not detected in liver cholangiocytes (HPA tissue IHC). Compare like cell types when judging intensity; a whole-section average can hide that variation (general IHC practice). |
| Strength of IHC evidence | The HPA tissue pattern is Supported, with medium consistency against RNA expression and supporting external characterisation (HPA tissue IHC). HPA038002 has Supported IHC status (HPA antibodies). These ratings support the reported pattern without making every unexpected stained cell specific. |
| IF/ICC: where should signal appear? | Expect nucleoplasmic signal: HPA lists the nucleoplasm as its supported main location and HPA038002 as Supported for ICC (HPA subcellular; HPA antibodies). This localisation answers the IF/ICC interpretation question; the application has its own guide page. |
| Fixation sensitivity | Target-specific effects of fixation on METTL14 staining are unreported in the supplied UniProt and HPA records. Do not infer a retrieval requirement or a changed positive-cell pattern from the nuclear annotation or HPA staining levels. |
| Situation | Likely cause | Next action |
|---|---|---|
| High-staining reference cells show no nuclear colour. | The expected cell population may be absent from the field, or the IHC run may have failed (general IHC practice); HPA reports high staining only for the specified alveolar type I cells and pachytene spermatocytes (HPA tissue IHC). | Verify cell identity and section quality, then review run controls and each staining step (general IHC practice). Avoid assigning a METTL14-specific fixation cause from these sources. |
| Colour is mainly cytoplasmic while nuclei remain pale. | The distribution disagrees with the nuclear METTL14 annotation and HPA nuclear tissue profile (UniProt Q9HCE5; HPA tissue IHC). Nonspecific staining or background is possible (general IHC practice). | Check negative-control staining and whether the chromogen follows cell outlines or tissue deposits; use the counterstain to identify nuclei (general IHC practice). |
| Liver cholangiocytes stain strongly. | HPA lists cholangiocytes as not detected in its liver IHC observations (HPA tissue IHC). Cross-reactivity or endogenous detection activity is possible, especially if colour is extranuclear (general IHC practice). | Confirm the cells are cholangiocytes and inspect a negative control; interpret any reproducible nuclear discrepancy in light of the HPA observation, without assuming the HPA category is an absolute biological rule. |
| Broad brown haze makes nuclei hard to score. | Excess background, residual endogenous detection activity, or precipitated chromogen can obscure localisation (general IHC practice). Haze is not the distinct nuclear pattern reported for METTL14 (HPA tissue IHC). | Inspect cell-free regions and the negative control; review blocking, washes, and chromogen development within the established IHC workflow (general IHC practice). |
| A low-staining cell population appears negative. | Some HPA-listed populations have low staining, including hippocampal neuronal cells and cardiomyocytes (HPA tissue IHC). Weak signal may be difficult to distinguish from background in a given run (general IHC practice). | Check a high-staining reference population in the same run before calling the assay unsuccessful (HPA tissue IHC; general IHC practice). Record the cell type and detection limit when reporting a negative result. |
| IF/ICC signal appears outside the nucleus. | HPA reports supported nucleoplasmic localisation for METTL14 and Supported ICC status for HPA038002 (HPA subcellular; HPA antibodies). Extranuclear signal therefore needs scrutiny. | Compare the signal with the nuclear counterstain and appropriate negative control (general IF practice). Use the separate IF/ICC guide for assay setup; this IHC interpretation does not establish an IF/ICC protocol. |
Experiment evidence
IHC & IF images showing METTL14 staining
A07389-2 · IHCIHC analysis of METTL14 using anti-METTL14 antibody (A07389-2). METTL14 was detected in paraffin-embedded section of human breast cancer tis
A07389-2 · IFIF analysis of METTL14 using anti-METTL14 antibody (A07389-2). METTL14 was detected in immunocytochemical section of A431 cells. Enzyme anti
M07389 · IHCM07389 staining METTL14 in human testis tissue sections by Immunohistochemistry (IHC-P -paraformaldehyde-fixed, paraffin-embedded sections).
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000145388-METTL14/tissue/cerebral+cortex#imid_10114760
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000145388-METTL14/tissue/colon#imid_10114678
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000145388-METTL14/tissue/liver#imid_10114687
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000145388-METTL14/tissue/kidney#imid_10114688
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA038002; green = METTL14, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA038002; green = METTL14, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for METTL14 IHC & IF
🧪Run lung first: alveolar type I cells should show nuclear staining (HPA: High in alveolar cells type I; UniProt: Nucleus). Run liver as the negative tissue, scoring cholangiocytes (HPA: Not detected in cholangiocytes); on the lung slide, internal negative cells should lack nuclear DAB above background, although HPA does not identify a validated negative cell type there.
Positive control tissue: Lung (Alveolar cells type I, HPA High)
Negative control tissue: Liver (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show METTL14 in A-431, U-251MG, U2OS, with annotated localisation: Nucleoplasm (supported) (HPA subcellular).
Technical controls: Include a no-primary (secondary-only) slide, an isotype control matched to the rabbit primary antibody’s clonality and concentration, and METTL14-knockout tissue or a peptide-block control if the immunizing peptide is available (caption: rabbit anti-METTL14 antibody). Quench endogenous peroxidase and check for endogenous biotin in lung sections when using the caption’s biotin–streptavidin/DAB detection (caption: biotinylated secondary, SABC and DAB).
⚠️Feasibility: A target-specific fixation window and fixation effect are unreported; the selected A07389-2 tissue-IHC caption does not state a fixative. The demonstrated paraffin-section workflow uses heat retrieval in EDTA at pH 8.0 (caption: EDTA retrieval), but the evidence does not establish whether frozen sections or IF are easier. In lung, thin alveolar type I cells can make nuclear signal difficult to assign, so assess staining against the no-primary slide (HPA: High in alveolar cells type I; UniProt: Nucleus).
HPA tissue IHC evidence for METTL14
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Supported — Medium consistency between antibody staining and RNA expression data. External characterization data supports antibody staining.). 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 |
|---|---|---|---|---|
| Lung | Alveolar cells type I | High | Protein (IHC) | HPA → |
| Testis | Pachytene spermatocytes | High | Protein (IHC) | HPA → |
| Adipose tissue | Adipocytes | Medium | Protein (IHC) | HPA → |
| Appendix | Glandular cells | Medium | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Liver | Cholangiocytes | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced METTL14 IHC Tips
Troubleshoot chromogenic METTL14 IHC in paraffin sections by checking retrieval, nuclear localisation, controls and scoring before interpreting differences in staining.
What should I adjust when METTL14 staining is weak after antigen retrieval?
Use heat-mediated retrieval in EDTA at pH 8.0 before chromogenic IHC of paraffin sections (datasheet A07389-2). The selected tissue image used that retrieval method, followed by 2 μg/mL primary antibody overnight at 4°C (caption A07389-2). If staining remains weak, compare retrieval heating times on adjacent sections while holding antibody concentration and detection conditions constant; the caption does not specify a heating time (caption A07389-2; standard IHC practice). Check tissue preservation and confirm that any increased signal is nucleoplasmic rather than diffuse background (HPA: supported nucleoplasmic localisation; standard IHC practice). Record the chosen heating time for subsequent runs (standard IHC practice).
Could fixation explain inconsistent METTL14 staining across paraffin blocks?
The selected paraffin-section image does not state a fixative or fixation duration, so METTL14-specific fixation sensitivity is unknown (caption A07389-2). Compare blocks with documented processing histories and examine adjacent sections for preservation before changing the staining workflow (standard IHC practice). Keep retrieval at EDTA pH 8.0, primary antibody at 2 μg/mL overnight at 4°C, and detection conditions matched during that comparison (caption A07389-2; standard IHC practice). If staining differs, repeat on similarly processed sections and assess nuclear signal against background before attributing the difference to fixation (HPA: supported nucleoplasmic localisation; standard IHC practice).
How should I assess METTL14 staining outside nuclei?
Expect predominantly nuclear staining: UniProt places METTL14 in the nucleus, and HPA supports a nucleoplasmic location (UniProt Q9HCE5 subcellular location; HPA: nucleoplasm supported). Inspect the chromogenic signal against a nuclear counterstain so that crowded cells and overlapping cytoplasm do not obscure its compartment (standard IHC practice). Strong diffuse cytoplasmic or membrane staining warrants a review of blocking, antibody concentration and detection controls; METTL14 has no annotated transmembrane segment (UniProt Q9HCE5 topology; standard IHC practice). Compare suspect regions with well-preserved internal cells rather than interpreting localisation at torn edges or damaged tissue (standard IHC practice).
Can isoforms or epitope accessibility explain uneven nuclear staining?
The supplied UniProt record annotates no METTL14 isoforms, while describing a 456-residue chain and phosphorylation at serine 399 (UniProt Q9HCE5 record). The antibody epitope is unspecified, so neither isoform selection nor phosphorylation-dependent recognition can be assigned as the cause of uneven staining (record; caption A07389-2). Compare adjacent sections using the documented EDTA pH 8.0 retrieval and 2 μg/mL antibody conditions before altering one variable at a time (datasheet A07389-2; caption A07389-2; standard IHC practice). If a second antibody with a documented distinct epitope is available, concordant nuclear staining can strengthen an accessibility assessment (standard IHC practice).
How can I compare METTL14 IHC with a multiplex IF experiment?
Use IF as a separate optimisation: this selected antibody has a documented paraffin-section chromogenic workflow, while HPA reports nucleoplasmic IF localisation without establishing conditions for this SKU (caption A07389-2; HPA: nucleoplasm supported). Pair METTL14 with a validated marker for the expected cell type, such as pachytene spermatocytes when examining testis, and assess nuclear colocalisation at the single-cell level (HPA: high in pachytene spermatocytes; standard IF practice). Choose a fluorophore in a channel with low measured tissue autofluorescence and include single-channel controls (standard IF practice). METTL14 has no transmembrane segment, so optimise permeabilisation for access to its nucleoplasmic epitope without assuming a membrane-facing orientation (UniProt Q9HCE5 topology; HPA: nucleoplasm supported; standard IF practice).
What should I check when the DAB signal is widespread?
The selected IHC image used 10% goat serum blocking, a biotinylated secondary antibody and streptavidin-biotin detection with DAB (caption A07389-2). Include a no-primary control and review endogenous peroxidase blocking, since either nonspecific detection or residual enzyme activity can produce chromogenic background (standard IHC practice). If background follows the secondary detection system, assess its blocking and washing steps before changing the primary antibody conditions (standard IHC practice). Compare the result with expected nucleoplasmic staining and score only intact cells whose nuclei can be resolved against the counterstain (HPA: nucleoplasm supported; standard IHC practice).
How should I quantify METTL14 across sections with different cellularity? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and nuclear staining threshold before scoring, because METTL14 is reported as ubiquitously nuclear with cell-dependent staining levels (HPA: tissue IHC profile). For each comparable region, report the percentage of positive nuclei and an H-score based on nuclear intensity and percentage; record the number of evaluable cells (standard IHC practice). If counting positive cells per mm², also report total evaluable cells per mm² so differences in cellularity are visible (standard IHC practice). Normalise comparisons to the same cell type, sampled area, section quality and staining batch, and exclude damaged regions consistently (standard IHC practice).
Which patterns support genuine METTL14 staining rather than artefact?
A credible signal is nucleoplasmic in intact cells, consistent with UniProt nuclear localisation and HPA subcellular evidence (UniProt Q9HCE5 subcellular location; HPA: nucleoplasm supported). HPA reports high staining in lung alveolar type I cells and testis pachytene spermatocytes, but no detected staining in liver cholangiocytes; evaluate the identified cell type rather than the organ alone (HPA: tissue IHC). Treat edge-restricted staining, necrotic areas, diffuse signal outside nuclei and staining in no-primary controls as reasons to investigate artefact (HPA: nucleoplasm supported; standard IHC practice). Review peroxidase blocking and detection controls before interpreting DAB deposits as METTL14 expression (standard IHC practice).
Boster reagents
Best METTL14 / N(6)-adenosine-methyltransferase non-catalytic subunit METTL14 IHC Antibodies
A07389-2 and M07389 have human paraffin-section IHC images (catalog IHC captions). A07389-2 also has an IF image in A431 cells (catalog IF caption); M07389 lists human and mouse reactivity (catalog).
Real IHC data
Anti-METTL14 Antibody ®
Cat # A07389-2
Real IHC data
Anti-METTL14 Antibody (N-Term)
Cat # M07389
A07389-2 will render with human breast cancer paraffin-section IHC data and has IF data in A431 cells (A07389-2 IHC and IF captions). M07389 will render with IHC data from paraformaldehyde-fixed, paraffin-embedded human testis (M07389 IHC caption).
Which to pick: For human tissue IHC, A07389-2 has an image-documented paraffin-section result and a 2–5 μg/mL IHC range; its IHC caption does not report the fixative (A07389-2 IHC caption; catalog). For IF/ICC, choose A07389-2 because its IF image documents A431 cells at 5 μg/mL (A07389-2 IF caption; catalog). For human or mouse tissue IHC, M07389 lists both species and IHC-P, though its 1:250 paraformaldehyde-fixed paraffin-section image documents human testis only (M07389 catalog; M07389 IHC caption).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q9HCE5 (MET14_HUMAN, N(6)-adenosine-methyltransferase non-catalytic subunit METTL14).
- Human Protein Atlas. METTL14 tissue IHC expression (reliability: Supported).
- Human Protein Atlas. METTL14 subcellular location (ICC-IF): Localized to the nucleoplasm..
- Human Protein Atlas. METTL14 antibody validation summary (1 antibodies).
- Association of METTL14 expression with prognosis and immunotherapy in breast cancer. Translational cancer research 2026 — PMC12885814.
- METTL14 regulate LRIG1 expression via m6A to affect nucleus pulposus cell senescence in intervertebral disc degeneration. Scientific reports 2026 — PMC13197395.
- Methyltransferase-like 14 promotes the tumorigenesis and proliferation of pancreatic cancer cells through myc proto-oncogene signaling pathway. CytoJournal 2024 — PMC11683408.
- METTL14 attenuates cancer stemness by suppressing ATF5/WDR74/β-catenin axis in gastric cancer. Cancer science 2025 — PMC11711053.
- PubMed PMID:10997877 — UniProt-cited evidence.
- PubMed PMID:12168954 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for METTL14 (N(6)-adenosine-methyltransferase non-catalytic subunit METTL14). Expected localisation Nuclear staining in tissue (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A07389-2). Validated IHC antibody: A07389-2 at 2-5μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Lung (Alveolar cells type I); negative Liver. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human breast cancer tissue; fixative not specified (datasheet A07389-2); 2. Fixation: Image fixative and duration unreported (datasheet A07389-2); 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: EDTA pH 8.0 (datasheet A07389-2); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A07389-2); 8. Primary antibody: Rabbit anti-METTL14, 2-5μg/ml (datasheet A07389-2); 9. Primary incubation: Overnight at 4 °C (datasheet A07389-2). Watch for: Fixation: Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A07389-2); Caveat: Liver cholangiocytes may show no detectable staining (HPA tissue IHC); Isoform / epitope: 0 annotated isoforms; one chain, residues 1–456 (UniProt); Regulation: Low tissue specificity; no regulator reported (HPA tissue IHC). Evidence: UniProt Q9HCE5, HPA tissue IHC · Supported, HPA ICC-IF · nucleoplasm., Datasheet A07389-2, PMC12885814, PMC13197395. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/METTL14/immunohistochemistry-ihc-guide.