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KMT2A / Histone-lysine N-methyltransferase 2A · IHC design guide
Design Immunohistochemistry for KMT2A
Plan chromogenic KMT2A IHC in paraffin sections around the observed nuclear tissue pattern (HPA tissue IHC). This guide covers fixation consistency, staining assessment, and catalog IHC dilution ranges of 1:100–1:300 for A00402 and 1:200–1:1000 for M00402 (datasheets).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
KMT2A 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 across tissues (HPA tissue IHC) | |
| Staining pattern | Nuclei across tissue cell types (HPA tissue IHC) | |
| Antigen retrieval | Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen) | |
| Positive control | Placenta+4 more · see all | |
| Negative control | None in HPA (detected in all 45 tissues); use no-primary + isotype controls |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep formalin fixation consistent across paraffin sections. (standard IHC practice; not target-specific) | |
| Caveat | Staining and RNA show medium consistency (HPA tissue IHC) | |
| Regulation | Low tissue specificity (HPA tissue RNA) | |
| Isoform / epitope | 3 isoforms; N320/C180 cleavage makes epitope position relevant (UniProt) |
Section 1
Recommended KMT2A IHC & IF Protocols
Compare the catalog antibody’s IHC-P protocol with 4 published KMT2A IHC methods (PMC11782969; PMC8133758; PMC5199165; PMC7288919).
| Sample | Paraffin-embedded human lung-cancer tissue; fixative not specified (datasheet A00402) |
| Fixation | Image fixative and duration unreported (datasheet A00402); 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 Tris-EDTA buffer, pH 9.0, 20 min at 95–98 °C (standard rule: nuclear 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-KMT2A, 1:100-1:300 (datasheet A00402) |
| 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 | KMT2A-positive staining in endothelial cells of placenta (HPA tissue IHC: High). HPA tissue profile: Ubiquitous nuclear expression. No signal in the no-primary control. |
💡Decision noteStart with Tris-EDTA pH 9.0 retrieval at 95–98°C for 20 min (page antigen retrieval; UniProt Q03164: nucleus). Published methods also report citrate retrieval (PMC8133758; PMC7288919).
Section 2
What Is the Expected KMT2A Staining Pattern?
KMT2A should appear predominantly in nuclei across many cell types in paraffin sections (UniProt Q03164: nucleus; HPA: ubiquitous nuclear expression). Strong reference staining occurs in placental endothelial cells and testicular pachytene spermatocytes (HPA: High in both). KMT2A has no transmembrane segment (UniProt Q03164: topology). HPA rates the tissue IHC evidence Enhanced, while reporting medium consistency between antibody staining and RNA expression (HPA: reliability).
| Nuclear chromogen in placental endothelial cells or testicular pachytene spermatocytes, with identifiable tissue morphology. | This matches the expected compartment and two High reference cell populations (UniProt Q03164: nucleus; HPA: High in both). Compare nuclei within the same section and confirm that the counterstain still permits cell identification (general IHC practice). |
| Strong staining is confined to cytoplasm, with little or no nuclear signal. | Treat a cytoplasm-only IHC pattern as suspect because the established tissue profile is nuclear (HPA: ubiquitous nuclear expression). Review localization in a known-positive section and check the detection controls before assigning biological meaning (general IHC practice). |
| Intense staining appears in cells expected to show low signal, or in cells whose identity is uncertain. | HPA reports low staining in caudate glial cells, parathyroid glandular cells, liver cholangiocytes and adipocytes (HPA: Low in each). Verify cell identity and compare a no-primary control; unexpected chromogen can reflect cross-reactivity or endogenous detection activity (general IHC practice). |
| Brown color spreads across tissue structures or covers nuclei and surrounding areas uniformly. | A diffuse deposit cannot reliably establish nuclear localization (general IHC practice). Compare a no-primary control and inspect blocking, washing and detection conditions; widespread color may be assay background rather than KMT2A staining (general IHC practice). |
| No nuclear signal appears in a known-positive reference section. | Absence in placental endothelial cells or testicular pachytene spermatocytes conflicts with their High HPA staining (HPA: High in both). Check tissue integrity, antigen retrieval, primary antibody use and detection performance before interpreting a test section as negative (general IHC practice). |
💡Expected KMT2A appearanceCall a result positive when chromogen is predominantly nuclear in identifiable cells, with High signal in placental endothelial cells or pachytene spermatocytes; isolated cytoplasmic color or diffuse tissue-wide deposit is suspect (HPA: tissue IHC profile; UniProt Q03164: nucleus; general IHC practice).
| Reference cell population | Placental endothelial cells and testicular pachytene spermatocytes are High; bone marrow hematopoietic cells and bronchial respiratory epithelial cells are Medium (HPA: tissue IHC). Choose the comparison cell type before judging intensity. |
| Low staining is not absence | HPA lists low staining in glial cells of the caudate, cholangiocytes and adipocytes (HPA: tissue IHC). A weak result in these cells is a poor stand-alone failure criterion; compare a High reference population. |
| Protein processing and antibody epitope | KMT2A yields N320 and C180 cleavage products (UniProt Q03164: processing). Which products an antibody detects depends on its epitope; the supplied HPA validation entries do not specify an epitope, so do not infer fragment-specific staining. |
| Antibody validation | Two listed antibodies have Enhanced IHC status, CAB017794 and CAB024270 (HPA: antibodies). Enhanced means agreement from independent antibodies or orthogonal data (HPA: validation definition); it does not resolve every unexpected cell-level signal. |
| IF/ICC Q&A: Where should signal appear? | A: Mainly in the nucleoplasm (HPA: supported ICC-IF location). HPA also lists cytosol as an uncertain additional location (HPA: subcellular); this does not establish a cytoplasm-only IHC pattern. |
| Situation | Likely cause | Next action |
|---|---|---|
| Known-positive section lacks nuclear staining. | A failed staining run is possible when cells reported High are blank (HPA: placental endothelial cells; testicular pachytene spermatocytes). | Check section quality, antigen retrieval settings, primary antibody application and detection reagents against the run controls (general IHC practice). |
| Test section looks negative beside a strongly stained reference. | The tested cell population may normally stain less strongly; HPA reports both Medium and Low populations (HPA: tissue IHC). | Identify the cells being scored, retain the High reference for run acceptance, and interpret weak staining relative to the appropriate population (HPA: tissue IHC; general IHC practice). |
| Signal is mainly cytoplasmic. | The pattern conflicts with the established nuclear tissue profile (HPA: ubiquitous nuclear expression; UniProt Q03164: nucleus). | Recheck morphology and a known-positive nuclear control; assess no-primary background before accepting the staining as specific (general IHC practice). |
| Diffuse chromogen obscures nuclei. | Excess background or endogenous detection activity may obscure localization (general IHC practice). | Inspect a no-primary control, then review blocking, washing and chromogen development conditions; score only cells with interpretable nuclei (general IHC practice). |
| Unexpectedly strong staining appears in a low-staining population. | Cell misidentification, cross-reactivity or endogenous detection activity are possibilities (HPA: Low populations; general IHC practice). | Confirm cell morphology and examine a no-primary control; compare the pattern with a High reference section before assigning positivity (HPA: High populations; general IHC practice). |
| Results differ between antibodies. | The listed antibodies have different application validation records, and the supplied entries do not establish matching epitopes (HPA: antibodies). | Check each antibody’s IHC validation and documented epitope, if available; compare nuclear localization and the same reference cell populations (HPA: tissue IHC; general IHC practice). |
Experiment evidence
IHC & IF images showing KMT2A staining
A00402 · IHCImmunohistochemical analysis of paraffin-embedded human-lung-cancer, antibody was diluted at 1:200
A00402 · IHCImmunohistochemical analysis of paraffin-embedded human-lung-cancer, antibody was diluted at 1:200
M00402 · IHCImmunohistochemistry validation of KMT2A using Anti-MLL KMT2A Monoclonal Antibody (M00402). Immunohistochemistry (IHC) analysis of paraffin-
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000118058-KMT2A/tissue/cerebral+cortex#imid_18126523
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000118058-KMT2A/tissue/colon#imid_18126449
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000118058-KMT2A/tissue/liver#imid_18126442
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000118058-KMT2A/tissue/kidney#imid_18126451
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA044910; green = KMT2A, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA044910; green = KMT2A, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for KMT2A IHC & IF
🧪Run placenta first and assess endothelial cells for nuclear staining (HPA: High in placental endothelial cells; UniProt Q03164: nucleus). HPA detects KMT2A in all 45 scored tissues, so there is no supported negative tissue; use no-primary and isotype controls, in which cells on the positive slide should show only background staining (HPA: no negative tissue; standard IHC practice).
Positive control tissue: Placenta (Endothelial cells, HPA High)
Negative control tissue: None in HPA: KMT2A is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead.
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show KMT2A in A-431, U-251MG, U2OS, KOLF2.1J, NIH 3T3, with annotated localisation: Nucleoplasm (supported) (HPA subcellular).
Technical controls: Include a secondary-only slide, a concentration-matched isotype control matched to the primary antibody’s host species and isotype, and KMT2A-knockout material if available as a biological negative (standard IHC practice). For chromogenic placenta IHC, block endogenous peroxidase and check blood-rich areas for background signal (standard IHC practice).
⚠️Feasibility: No target-specific fixation window or fixation effect is reported in the supplied evidence; the A00402 paraffin-section caption gives 1:200 but leaves the fixative unreported (A00402 caption). Antigen-retrieval dependency is unreported, so optimize retrieval on control sections; the supplied evidence does not establish that frozen sections or IF are easier (A00402 caption; HPA: ICC-IF images). For placenta, blood-associated peroxidase background can complicate chromogenic interpretation (standard IHC practice).
HPA tissue IHC evidence for KMT2A
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — Medium consistency between antibody staining and RNA expression data.). Rows are taken directly from the HPA tissue chart — click any row's HPA link to view the source.
Positive expression · recommended positive controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Placenta | Endothelial cells | High | Protein (IHC) | HPA → |
| Testis | Pachytene spermatocytes | High | Protein (IHC) | HPA → |
| Adrenal gland | Glandular cells | 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 |
|---|---|---|---|---|
| None in HPA: KMT2A is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead. | ||||
Section 3
Advanced KMT2A IHC Tips
Troubleshoot KMT2A staining in paraffin sections by checking nuclear localisation, retrieval, antibody epitope, detection controls, and scoring consistency (UniProt Q03164; HPA tissue IHC).
What should I adjust if KMT2A nuclear staining is weak after retrieval?
Use Tris-EDTA pH 9.0 HIER at 95–98 °C for 20 min (page retrieval specification). Keep section thickness and cooling conditions consistent while comparing runs, since both can change staining intensity (standard IHC practice). If staining remains weak, compare a carefully controlled alternative retrieval buffer or heating time on adjacent sections, recording tissue damage alongside nuclear signal (standard IHC practice). The catalog antibody has a paraffin-section image at 1:200, but its caption does not report retrieval conditions (A00402 caption). Judge any adjustment by retained morphology and nuclear staining in an appropriate positive control (UniProt Q03164 localisation; standard IHC practice).
How can I assess whether fixation caused weak or uneven KMT2A staining?
KMT2A-specific sensitivity to fixative type or fixation duration is unknown from the supplied evidence (A00402 caption; HPA tissue IHC). The catalog image identifies paraffin-embedded human lung cancer and 1:200 antibody dilution, but does not state a fixative (A00402 caption). Record fixation and processing history, then compare sections processed alike before attributing a staining difference to antigen abundance (standard IHC practice). Examine nuclear staining and morphology in an internal or parallel positive control, while checking whether weak areas coincide with poor preservation (UniProt Q03164 localisation; standard IHC practice). Do not infer fixation tolerance from KMT2A topology, modifications, or tissue staining patterns (UniProt Q03164; HPA tissue IHC).
What staining pattern should I expect for KMT2A in paraffin sections?
Prioritise nuclear staining: KMT2A is assigned to the nucleus, and tissue IHC describes ubiquitous nuclear expression (UniProt Q03164 localisation; HPA tissue IHC). Its pattern may appear diffuse when it is not associated with cleavage product N320, so uniform nuclear signal can be plausible (UniProt Q03164 localisation). Supported nucleoplasmic localisation in cell imaging reinforces the nuclear expectation, while its additional cytosolic localisation is uncertain (HPA subcellular). Compare nuclear signal with a counterstain and assess the same cell type across matched sections (standard IHC practice). Predominantly cytoplasmic staining warrants checks of retrieval, antibody dilution, and detection background before a biological interpretation (UniProt Q03164 localisation; standard IHC practice).
Could KMT2A processing or isoforms explain discordant IHC staining?
Check the antibody’s stated immunogen or epitope before comparing staining across KMT2A assays, because the record lists 3 isoforms (UniProt Q03164 isoforms). KMT2A is processed into N320, residues 1–2718, and C180, residues 2719–3969 (UniProt Q03164 processing). An antibody recognising one fragment may report a different distribution or abundance from an antibody recognising the other; confirm the actual epitope before making that inference (UniProt Q03164 processing; standard IHC interpretation). The SET domain lies at residues 3829–3945, within C180, which can help map a disclosed epitope (UniProt Q03164 domains and processing). Compare localisation and control staining rather than treating disagreement between unknown epitopes as proof of isoform change (standard IHC practice).
How should I check a KMT2A IHC result with multiplex immunofluorescence?
Use IF/ICC as a separate assay and compare nuclear KMT2A signal with a marker identifying the cell population of interest (UniProt Q03164 localisation; standard IF practice). Choose spectrally separated fluorophores and place the weaker expected signal in a channel with low tissue autofluorescence (standard IF practice). For an intracellular nuclear epitope, optimise permeabilisation so antibody can reach the nucleus while preserving morphology (UniProt Q03164 localisation; standard IF practice). Include single-stain and no-primary controls to assess bleed-through and background before interpreting overlap (standard IF practice). Cell-image evidence supports nucleoplasmic localisation, but does not establish fixation or permeabilisation settings for a particular antibody (HPA subcellular).
How do I distinguish KMT2A staining from chromogenic background?
Inspect no-primary and detection-only controls for nonspecific chromogen deposition before adjusting the primary antibody (standard IHC practice). Include an appropriate peroxidase block when using peroxidase-based DAB detection, and check whether residual signal persists without primary antibody (standard IHC practice). Titrate the catalog antibody around its captioned 1:200 dilution while holding retrieval and detection conditions constant (A00402 caption; standard IHC practice). Evaluate staining against nuclei identified by the counterstain, since nuclear expression is the principal tissue pattern (HPA tissue IHC; standard IHC practice). Diffuse extracellular colour, tissue-edge accentuation, or signal confined to damaged areas should prompt a background review (standard IHC practice).
How should I score KMT2A staining across comparable paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and score nuclear staining separately from cytoplasmic colour, reflecting the reported predominant nuclear pattern (HPA tissue IHC; standard IHC practice). Report an H-score using nuclear intensity categories 0–3 and each category’s percentage of cells, or report the percentage of nuclear-positive cells (standard IHC practice). For spatial comparisons, count positive nuclei per mm² of viable tissue and state the analysed area (standard IHC practice). Normalise counts to all evaluable nuclei in the same cell population, and apply one threshold and image-acquisition setting across cases (standard IHC practice). Record excluded necrotic or damaged regions so apparent changes in staining are interpretable (standard IHC practice).
When is an apparent KMT2A-positive result convincing?
A convincing result shows reproducible nuclear staining in intact cells, consistent with the reported nuclear localisation and ubiquitous nuclear tissue pattern (UniProt Q03164 localisation; HPA tissue IHC). Examine the stained cell type explicitly: high staining is reported in placental endothelial cells and testicular pachytene spermatocytes, while tissue signals vary elsewhere (HPA tissue IHC). Predominantly cytoplasmic colour deserves caution because additional cytosolic localisation has uncertain support in cell imaging (HPA subcellular). Exclude section-edge accentuation, necrotic areas, and staining retained in no-primary or peroxidase controls before calling cells positive (standard IHC practice). Interpret intensity alongside morphology, controls, and the antibody’s known epitope when available (standard IHC practice; UniProt Q03164 processing).
Boster reagents
Best KMT2A / Histone-lysine N-methyltransferase 2A IHC Antibodies
Two anti-KMT2A antibodies have paraffin-section IHC images (catalog IHC captions); both list IF, but no IF images are supplied (catalog applications; catalog IF image lists).
Real IHC data
Anti-MLL KMT2A Antibody
Cat # A00402
Real IHC data
Anti-MLL KMT2A Monoclonal Antibody
Cat # M00402
A00402 lists IHC and IF for human, mouse and rat; its IHC captions show paraffin-embedded human lung cancer and brain (catalog applications; catalog reactivity; A00402 IHC captions). M00402 lists IHC and IF for human samples; its IHC caption shows paraffin-embedded human lung and esophagus cancers (catalog applications; catalog reactivity; M00402 IHC caption).
Which to pick: For human tissue IHC, either SKU has a paraffin-section example: A00402 in lung cancer and brain, and M00402 in lung and esophagus cancers (A00402 IHC captions; M00402 IHC caption). For IF/ICC, both list IF, but neither has an IF image in the payload; A00402 is a rabbit antibody and M00402 is a mouse monoclonal, so choose based on the planned detection system (catalog applications; catalog IF image lists; catalog host and clone fields). For mouse or rat samples, choose A00402 because its reactivity list includes both, whereas M00402 lists human only; the captions report paraffin embedding but do not report the fixative (catalog reactivity; A00402 IHC captions; M00402 IHC caption).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q03164 (KMT2A_HUMAN, Histone-lysine N-methyltransferase 2A).
- Human Protein Atlas. KMT2A tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. KMT2A subcellular location (ICC-IF): Mainly localized to the nucleoplasm. In addition localized to the cytosol..
- Human Protein Atlas. KMT2A antibody validation summary (3 antibodies).
- Histone methyltransferase KMT2A promotes pulmonary fibrogenesis via targeting pro-fibrotic factor PU.1 in fibroblasts. Clinical and translational medicine 2025 — PMC11782969.
- Genome-scale CRISPR-Cas9 screen of Wnt/β-catenin signaling identifies therapeutic targets for colorectal cancer. Science advances 2021 — PMC8133758.
- Kmt2a cooperates with menin to suppress tumorigenesis in mouse pancreatic islets. Cancer biology & therapy 2016 — PMC5199165.
- KMT2A regulates cervical cancer cell growth through targeting VDAC1. Aging 2020 — PMC7288919.
- PubMed PMID:1423624 — UniProt-cited evidence.
- PubMed PMID:8703835 — UniProt-cited evidence.
- PubMed PMID:16554811 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for KMT2A (Histone-lysine N-methyltransferase 2A). Expected localisation Nuclear staining across tissues (HPA tissue IHC). Antigen retrieval: Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen). Validated IHC antibody: A00402 at 1:100-1:300 (the product's own IHC validation image is shown). Controls (HPA): positive Placenta (Endothelial cells); negative None in HPA (detected in all 45 tissues); use no-primary + isotype controls. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Paraffin-embedded human lung-cancer tissue; fixative not specified (datasheet A00402); 2. Fixation: Image fixative and duration unreported (datasheet A00402); 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 Tris-EDTA buffer, pH 9.0 , 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-KMT2A, 1:100-1:300 (datasheet A00402); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep formalin fixation consistent across paraffin sections. (standard IHC practice; not target-specific); Caveat: Staining and RNA show medium consistency (HPA tissue IHC); Isoform / epitope: 3 isoforms; N320/C180 cleavage makes epitope position relevant (UniProt); Regulation: Low tissue specificity (HPA tissue RNA). Evidence: UniProt Q03164, HPA tissue IHC · Enhanced, HPA ICC-IF · Mainly localized to the nucleoplasm. In addition localized to the…, Datasheet A00402, PMC11782969, PMC8133758. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/KMT2A/immunohistochemistry-ihc-guide.