METTL14 / N(6)-adenosine-methyltransferase non-catalytic subunit METTL14 · IHC design guide

Design Immunohistochemistry for METTL14

Plan chromogenic IHC for METTL14 in paraffin sections using the catalog antibody at 2–5 μg/ml (datasheet A07389-2). Expect widespread nuclear staining, with high signal in lung alveolar type I cells and testis pachytene spermatocytes (HPA tissue IHC).

Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
Immunohistochemistry protocol sheet for METTL14 (IHC for METTL14): expected localisation Nuclear staining in tissue sections (HPA tissue IHC), antibody A07389-2, validated IHC image, and IHC protocol steps
Printable METTL14 IHC protocol sheet — expected localisation Nuclear staining in tissue sections (HPA tissue IHC), antibody A07389-2, controls and protocol steps. Open the full METTL14 IHC guide →

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 sections (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 Keep fixation consistent across sections (standard IHC practice; not target-specific)
Caveat Cholangiocytes may lack detectable signal (HPA tissue IHC)
Regulation High in pachytene spermatocytes (HPA tissue IHC)
Isoform / epitope No isoforms or processing annotated; no TM segment (UniProt)
Section 1

Recommended METTL14 IHC & IF Protocols

The catalog antibody’s IHC protocol uses EDTA pH 8.0 retrieval (datasheet A07389-2). The published protocols below provide three tissue-specific comparisons (PMC12885814; PMC13197395; PMC11711053).

Recommended immunohistochemistry (IHC-P) protocol parameters
SampleParaffin-embedded human breast cancer tissue; fixative not specified (datasheet A07389-2)
FixationImage fixative and duration unreported (datasheet A07389-2); verify before use.
Sectioning4–5 µm sections on charged slides (standard)
DeparaffinisationXylene, graded ethanol series to water (standard)
Antigen retrievalHeat retrieval: EDTA pH 8.0 (datasheet A07389-2); 20 min, 95–100 °C (standard)
Peroxidase block3% H2O2, 10 min, room temperature (standard)
Blocking10% goat serum (datasheet A07389-2)
Primary antibodyRabbit anti-METTL14, 2-5μg/ml (datasheet A07389-2)
Primary incubationOvernight at 4 °C (datasheet A07389-2)
DetectionStreptavidin-biotin complex (SABC), DAB chromogen (datasheet A07389-2)
CounterstainHematoxylin, blue, dehydrate and mount (standard)
Expected resultMETTL14-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 EDTA pH 8.0 for the catalog antibody (datasheet A07389-2); the published protocols used EDTA pH 9.0 or citrate pH 6.0 with different antibodies (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, with particularly strong staining reported in lung alveolar type I cells and testis pachytene spermatocytes (HPA tissue IHC). HPA describes the tissue pattern as ubiquitous nuclear expression and rates its antibody evidence Supported, with medium consistency between staining and RNA data (HPA tissue IHC).

What am I looking at on my slide?
Distinct nuclear staining in alveolar type I cells or pachytene spermatocytes, with other nuclei also stained.This fits the reported high signal in those two cell populations and the broader nuclear pattern (HPA tissue IHC). Judge the named cells within their tissue context: HPA's High level describes their observed staining, not every cell in the section. Nuclear localization also agrees with UniProt Q9HCE5.
Predominantly cytoplasmic, membrane-like, or extracellular staining, with little nuclear signal.That distribution conflicts with the nuclear assignment (UniProt Q9HCE5; HPA tissue IHC). Treat it as a warning for nonspecific staining or a detection artefact, rather than evidence for a new METTL14 compartment. Recheck the slide against a known nuclear-positive cell population and the negative controls (standard IHC practice).
Strong chromogenic signal in liver cholangiocytes, especially if nearby nuclei show no convincing stain.HPA reports METTL14 as Not detected in cholangiocytes (HPA tissue IHC). Unexpected signal there raises concern for cross-reactivity or endogenous detection activity, but the HPA observation alone cannot identify which. Compare its compartment and distribution with a positive tissue and appropriate reagent controls (standard IHC practice).
A diffuse haze covers nuclei, cytoplasm, and spaces between cells; individual stained nuclei are hard to distinguish.This is difficult to score as METTL14 because the expected localization is nuclear (UniProt Q9HCE5; HPA tissue IHC). Broad background may reflect detection reagents, insufficient blocking, or excessive staining conditions (standard IHC practice). Assess controls before interpreting small intensity differences between cell populations.
No nuclear signal in alveolar type I cells or pachytene spermatocytes on an otherwise assessable section.Those populations are reported High in HPA tissue IHC, so a blank result calls for a technical check. Their reported staining does not guarantee that every specimen will match the reference. Verify tissue identity and preservation, the antibody and detection steps, and an independent positive control before calling the sample negative (standard IHC practice).
💡Expected METTL14 appearanceCall a result positive when nuclei are clearly stained, especially in HPA High alveolar type I cells or pachytene spermatocytes; predominant cytoplasmic or diffuse signal is suspect (HPA tissue IHC; UniProt Q9HCE5).
How each factor affects the staining
Cell population and tissue contextHPA reports High staining in lung alveolar type I cells and testis pachytene spermatocytes, Medium staining in several other listed populations, and no detected staining in liver cholangiocytes (HPA tissue IHC). Select and score the named cells rather than assigning one intensity to a whole organ.
Compartment and protein topologyUniProt places METTL14 in the nucleus and reports no transmembrane segment or signal peptide (UniProt Q9HCE5). A membrane outline or secreted-looking deposit therefore lacks support as the expected pattern. HPA's nucleoplasmic ICC-IF localization offers a compartment cross-check (HPA ICC-IF).
Strength of reference evidenceThe tissue IHC profile is Supported, with medium consistency between antibody staining and RNA expression; HPA038002 is IHC Supported (HPA tissue IHC; HPA antibodies). Use the profile as a reference pattern, while investigating discordant results rather than treating its intensity labels as specimen-wide cutoffs.
IF/ICC cross-check: where should fluorescence appear?HPA reports supported nucleoplasmic localization and ICC-IF images from A-431, U-251MG, and U2OS cells (HPA ICC-IF). Expect a nucleoplasmic pattern when comparing localization across methods. IF/ICC preparation and detection belong to the separate IF/ICC guide.
Why is my staining missing, weak or wrong?
SituationLikely causeNext action
The known-positive nuclei are blank.A missing detection step, unsuitable reagent condition, or poor tissue preservation can suppress visible staining (standard IHC practice). HPA reports High staining in alveolar type I cells and pachytene spermatocytes (HPA tissue IHC).Confirm the named cells are present, inspect section quality, and check the catalog antibody's IHC-P instructions and the detection controls (standard IHC practice). Do not infer a METTL14-specific fixation effect: none is established by the supplied sources.
Signal is mostly outside nuclei.The observed compartment disagrees with nuclear METTL14 (UniProt Q9HCE5; HPA tissue IHC). Background or nonspecific binding is a possible explanation, not a diagnosis from appearance alone (standard IHC practice).Compare a known-positive tissue and negative reagent controls; assess nuclear and extranuclear staining separately (standard IHC practice). Treat extranuclear staining as unconfirmed unless independently supported.
Cholangiocytes stain strongly.HPA lists liver cholangiocytes as Not detected (HPA tissue IHC). Cross-reactivity or endogenous chromogenic activity may account for unexpected staining (standard IHC practice).Check the compartment, compare a known-positive population, and use suitable negative and endogenous-activity controls for the chosen detection system (standard IHC practice). Do not generalize the cholangiocyte result to every liver cell.
Background obscures nuclear boundaries.Widespread colour development can conceal the nuclear pattern expected for METTL14 (UniProt Q9HCE5; HPA tissue IHC). Excess primary or detection reagent, inadequate blocking, or residual endogenous activity are general IHC possibilities (standard IHC practice).Review reagent-only controls, blocking, antibody concentration, and development time under the chosen IHC-P workflow (standard IHC practice). Score intensity only where nuclei can be distinguished from surrounding background.
A specimen stains more weakly than the HPA example.HPA intensity levels are observations in specified cell populations; its tissue evidence has medium consistency with RNA expression (HPA tissue IHC). Cell mix, section quality, and assay conditions may complicate comparison (standard IHC practice).Compare the same cell type and compartment, include a known-positive section in the run, and document the observed nuclear intensity (standard IHC practice). Avoid assigning a universal positive cutoff from the HPA labels.
IF/ICC shows diffuse fluorescence instead of nucleoplasmic enrichment.HPA's supported ICC-IF location is the nucleoplasm (HPA ICC-IF). Diffuse fluorescence alone does not establish a different METTL14 location; background remains possible (standard IF practice).Compare with appropriate fluorescence controls and the HPA nucleoplasmic reference (HPA ICC-IF; standard IF practice). Use the separate IF/ICC guide for method choices; this IHC-P section supplies no IF protocol.

Sample controls for METTL14 IHC & IF

🧪Run testis first and look for nuclear staining in pachytene spermatocytes (HPA: High in pachytene spermatocytes; UniProt: Nucleus). Use liver cholangiocytes as the negative tissue comparator (HPA: Not detected in cholangiocytes); on the testis slide, treat cells without detectable nuclear signal as background comparators without assuming that other testicular cell types are METTL14-negative (HPA: High in pachytene spermatocytes).
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 control, a concentration-matched rabbit isotype control, and a METTL14 knockout section where available (caption: rabbit anti-METTL14 antibody; standard IHC controls). For chromogenic detection, quench endogenous peroxidase and check for endogenous biotin signal because the selected example uses a biotin-based system with DAB (caption: biotinylated secondary, SABC and DAB).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected A07389-2 paraffin-section caption does not state the fixative (caption: fixative not stated). The example uses heat retrieval in EDTA at pH 8.0, but does not establish that retrieval is required under every condition (caption: EDTA retrieval at pH 8.0). There is no supplied comparison showing that frozen sections or IF are easier; in testis, use nuclear morphology to identify pachytene spermatocytes and avoid scoring unrelated tubule cells as negatives solely from the HPA row (HPA: High in pachytene spermatocytes; 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

TissueCell typeLevelEvidenceSource
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

TissueCell typeLevelEvidenceSource
Liver Cholangiocytes Not detected Protein (IHC) HPA →
Section 3

Advanced METTL14 IHC Tips

Troubleshoot METTL14 staining in paraffin sections by checking retrieval, nuclear localisation, controls and cell-level scoring before interpreting chromogenic signal.

What retrieval should I use if METTL14 nuclear staining is weak?
Start with heat-mediated antigen retrieval in EDTA pH 8.0 for paraffin sections (datasheet A07389-2). Run a positive control alongside the test section, then compare nuclear signal and tissue damage before changing retrieval conditions (standard IHC practice). If staining remains weak, adjust heating time in small increments while holding antibody concentration and detection conditions constant (standard IHC practice). The reported image used 2 µg/mL primary antibody overnight at 4°C, followed by a biotinylated secondary antibody and DAB detection (datasheet A07389-2). Judge improvement by nuclear staining in intact cells, since METTL14 is nuclear (UniProt Q9HCE5; HPA: ubiquitous nuclear expression).
How can I troubleshoot METTL14 staining when fixation varies between specimens?
The selected paraffin-section caption does not state a fixative, so target-specific fixation sensitivity is unknown (datasheet A07389-2). Record each specimen’s fixation history and compare specimens processed under matched conditions before attributing intensity differences to METTL14 abundance (standard IHC practice). Where material permits, stain sections from differently processed blocks in the same run using EDTA pH 8.0 retrieval and identical detection settings (datasheet A07389-2; standard IHC practice). Examine nuclear preservation, morphology and nonspecific staining together, because damaged sections can undermine cell-level interpretation (standard IHC practice). Do not infer fixation tolerance from tissue-expression patterns or the protein’s lack of a transmembrane segment (HPA: tissue IHC profile; UniProt Q9HCE5 topology).
Should METTL14 staining appear in nuclei or cytoplasm in paraffin sections?
Score METTL14 primarily in nuclei: UniProt assigns it to the nucleus, and HPA reports supported nucleoplasmic localisation (UniProt Q9HCE5; HPA: subcellular). HPA also describes ubiquitous nuclear expression across tissues, although its tissue-IHC reliability is Supported with medium staining-to-RNA consistency (HPA: tissue IHC). Use a counterstain and sufficient magnification to distinguish nuclear DAB from pigment or signal overlying adjacent cytoplasm (standard IHC practice). If staining is predominantly cytoplasmic, repeat with a matched negative control and inspect retrieval, secondary-antibody background and tissue integrity before scoring it as METTL14 (standard IHC practice; UniProt Q9HCE5 localisation). Report nuclear and extranuclear observations separately rather than combining them into one intensity score (standard IHC practice).
Could an isoform or epitope explain inconsistent METTL14 IHC staining?
The supplied UniProt record lists 0 isoforms and one chain spanning residues 1–456; it does not establish where this antibody binds (UniProt Q9HCE5). A phosphoserine at residue 399 is annotated, but its effect on this antibody’s epitope is unknown (UniProt Q9HCE5; datasheet A07389-2: epitope not stated). Compare serial sections under the same EDTA pH 8.0 retrieval conditions before assigning regional differences to epitope accessibility (datasheet A07389-2; standard IHC practice). If results remain discordant, seek documented epitope information and independently validated antibody evidence before proposing an isoform explanation (standard IHC practice). Preserve separate records of staining location, intensity and processing for each section (standard IHC practice).
How should I assess METTL14 by IF alongside chromogenic IHC?
Treat IF as a separate assay: the selected EDTA pH 8.0 retrieval and 2 µg/mL primary-antibody conditions document paraffin-section IHC only (datasheet A07389-2). For multiplex IF, pair METTL14 with a validated marker of the expected cell type and assess colocalisation within counterstained nuclei (standard IF practice; UniProt Q9HCE5 localisation). Choose fluorophores after checking each specimen’s autofluorescence and include single-channel controls to detect bleed-through (standard IF practice). If an IF fixation and permeabilisation workflow exposes intracellular epitopes, optimise permeabilisation for nuclear access; METTL14 has no transmembrane segment (standard IF practice; UniProt Q9HCE5 topology). HPA reports supported nucleoplasmic localisation in ICC/IF, which can guide compartment checks without validating this catalog antibody for IF (HPA: subcellular).
How do I separate METTL14 signal from chromogenic IHC background?
Compare the stained section with a primary-omission control and inspect whether brown signal aligns with intact nuclei (standard IHC practice; UniProt Q9HCE5 localisation). The selected image used 10% goat-serum blocking, a biotinylated goat anti-rabbit secondary antibody and SABC with DAB (datasheet A07389-2). For that detection design, check endogenous biotin and peroxidase activity with suitable controls, and use a peroxidase block as a general chromogenic-IHC step (standard IHC practice). Titrate primary antibody around the reported 2 µg/mL while keeping retrieval and development consistent (datasheet A07389-2; standard IHC practice). Diffuse staining outside nuclei that persists in controls should be recorded as background rather than scored as METTL14 (standard IHC practice; UniProt Q9HCE5 localisation).
What is a defensible way to quantify METTL14 in stained sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and tissue region before scoring, then quantify nuclear staining because METTL14 is nuclear (standard IHC practice; UniProt Q9HCE5). Record the percentage of positive nuclei and intensity categories; an H-score from 0–300 combines their distribution (standard IHC practice). For spatial comparisons, report positive nuclei per mm² of evaluable tissue and normalise counts to the corresponding cell population or sampled area (standard IHC practice). Apply identical thresholds, counterstain assessment and imaging settings across specimens, excluding folds, necrosis and damaged edges (standard IHC practice). Report compartment-specific results and controls, since HPA describes supported tissue staining with only medium consistency relative to RNA expression (HPA: tissue IHC).
When is a METTL14-positive IHC result credible rather than artefactual?
A credible result shows reproducible staining in intact nuclei, consistent with UniProt nuclear annotation and HPA’s supported nucleoplasmic localisation (UniProt Q9HCE5; HPA: subcellular). HPA reports high staining in lung alveolar type I cells and testis pachytene spermatocytes, while liver cholangiocytes were not detected; use these observations as context, not universal thresholds (HPA: tissue IHC). Scrutinise cytoplasmic-only staining, isolated cut edges, necrotic regions and signal reproduced in primary-omission controls (standard IHC practice; UniProt Q9HCE5 localisation). Check endogenous peroxidase activity when DAB signal appears in unexpected compartments, and review morphology before calling cells positive (standard IHC practice). Finally, staining measures detected protein distribution; METTL14’s role as the METTL3-associated RNA-binding scaffold does not itself establish methyltransferase activity in a section (UniProt Q9HCE5 function).
Boster reagents

Best METTL14 / N(6)-adenosine-methyltransferase non-catalytic subunit METTL14 IHC Antibodies

IHC examples cover human breast cancer and testis sections (catalog IHC captions); IF/ICC imagery covers A431 cells (A07389-2 IF caption). Listed reactivity includes human, mouse, rat and monkey (catalog reactivity).

Real IHC data IHC analysis of METTL14 using anti-METTL14 antibody (A07389-2). METTL14 was detected in paraffin-embedded section of human breast cancer tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2μg/ml rabbit anti-METTL14 Antibody (A07389-2) 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. Left: Negative Control Right: Immunohistochemistry analysis of paraffin-embedded human breast cancer tissue, using METTL14 (A07389-2) Antibody
Anti-METTL14 Antibody ®
Cat # A07389-2
Real IHC data M07389 staining METTL14 in human testis tissue sections by Immunohistochemistry (IHC-P -paraformaldehyde-fixed, paraffin-embedded sections). Samples were incubated with primary antibody (1/250) for 1 hours at room temperature. A undiluted biotinylated goat polyvalent antibody was used as the secondary antibody.
Anti-METTL14 Antibody (N-Term)
Cat # M07389

A07389-2 has IHC imagery from paraffin-embedded human breast cancer tissue and IF/ICC imagery from A431 cells (A07389-2 image captions). M07389 has IHC imagery from paraformaldehyde-fixed, paraffin-embedded human testis tissue (M07389 IHC caption).

Which to pick: For tissue IHC, choose A07389-2 for paraffin-embedded human breast cancer sections (A07389-2 IHC caption: 2 μg/ml; fixative unreported) or M07389 for paraformaldehyde-fixed, paraffin-embedded human testis sections (M07389 IHC caption: 1:250). For IF/ICC, A07389-2 has an A431 cell image and a listed 5 μg/ml dilution (A07389-2 IF caption and catalog dilution). For broader listed species reactivity, M07389-1 is a monoclonal antibody with IHC and IF/ICC applications and human, mouse, rat and monkey reactivity (M07389-1 catalog applications, clone and reactivity); the payload provides no IHC image for it (M07389-1 catalog image alts).

Each figure is that product's own IHC / IF validation image from its datasheet.

References

  1. UniProt Consortium. UniProt entry Q9HCE5 (MET14_HUMAN, N(6)-adenosine-methyltransferase non-catalytic subunit METTL14).
  2. Human Protein Atlas. METTL14 tissue IHC expression (reliability: Supported).
  3. Human Protein Atlas. METTL14 subcellular location (ICC-IF): Localized to the nucleoplasm..
  4. Human Protein Atlas. METTL14 antibody validation summary (1 antibodies).
  5. Association of METTL14 expression with prognosis and immunotherapy in breast cancer. Translational cancer research 2026 — PMC12885814.
  6. METTL14 regulate LRIG1 expression via m6A to affect nucleus pulposus cell senescence in intervertebral disc degeneration. Scientific reports 2026 — PMC13197395.
  7. Methyltransferase-like 14 promotes the tumorigenesis and proliferation of pancreatic cancer cells through myc proto-oncogene signaling pathway. CytoJournal 2024 — PMC11683408.
  8. METTL14 attenuates cancer stemness by suppressing ATF5/WDR74/β-catenin axis in gastric cancer. Cancer science 2025 — PMC11711053.
  9. PubMed PMID:10997877 — UniProt-cited evidence.
  10. PubMed PMID:12168954 — UniProt-cited evidence.
  11. PubMed PMID:14702039 — UniProt-cited evidence.