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DNMT3A / DNA (cytosine-5)-methyltransferase 3A · IHC design guide
Design Immunohistochemistry for DNMT3A
Plan DNMT3A chromogenic IHC in paraffin sections around its widespread nuclear tissue pattern (HPA tissue IHC). Start the catalog antibody at 2–5 μg/ml (datasheet A00136-2) and assess staining alongside the reported high signal in adrenal gland glandular cells (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
DNMT3A 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 A00136-2) | |
| Positive control | Adrenal gland+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 fixation conditions consistent across samples (standard IHC practice; not target-specific) | |
| Caveat | Bone marrow hematopoietic cells show low staining (HPA tissue IHC) | |
| Regulation | Higher expression in fetal tissues (UniProt) | |
| Isoform / epitope | 3 isoforms; verify epitope coverage (UniProt) |
Section 1
Recommended DNMT3A IHC & IF Protocols
The catalog antibody’s IHC-P protocol is accompanied by published DNMT3A methods for patient FFPE tissue, odontogenic specimens, and lymphoma tissue microarrays (PMC2829483; PMC8757744; PMC5922379).
| Sample | Paraffin-embedded human colon cancer tissue; fixative not specified (datasheet A00136-2) |
| Fixation | Image fixative and duration unreported (datasheet A00136-2); 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 A00136-2); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A00136-2) |
| Primary antibody | Rabbit anti-DNMT3A, 2-5 μg/ml (datasheet A00136-2) |
| Primary incubation | Overnight at 4 °C (datasheet A00136-2) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet A00136-2) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | DNMT3A-positive staining in glandular cells of adrenal gland (HPA tissue IHC: High). HPA tissue profile: Ubiquitous nuclear expression. No signal in the no-primary control. |
💡Decision noteStart with EDTA pH 8.0 heat retrieval for the catalog antibody (datasheet A00136-2); adjust retrieval when adapting a published antibody protocol (PMC2829483; PMC8757744).
Section 2
What Is the Expected DNMT3A Staining Pattern?
DNMT3A should appear predominantly in nuclei on paraffin tissue sections: HPA describes ubiquitous nuclear expression, with high staining in several specified cell types (HPA: tissue IHC, Approved; medium consistency with RNA). UniProt also lists chromosome and cytoplasm, including accumulation at pericentric heterochromatin (UniProt Q9Y6K1: subcellular location). DNMT3A has no transmembrane segment (UniProt Q9Y6K1: topology).
| Predominant nuclear staining in glandular cells of the adrenal gland, appendix, cervix, or duodenum. | This fits the reported DNMT3A distribution: each listed cell population has high tissue staining (HPA: tissue IHC). Compare nuclei with nearby tissue structures on the same section; high staining is a relative observation, not a required intensity for every nucleus (HPA: tissue IHC, Approved). |
| Cytoplasmic staining dominates while nuclei remain faint or unstained. | The distribution conflicts with HPA's ubiquitous nuclear IHC profile (HPA: tissue IHC). Review the staining and controls for artefact before interpreting it as DNMT3A. Cytoplasmic localization is also listed by UniProt, so cytoplasmic signal alone cannot establish cross-reactivity (UniProt Q9Y6K1: subcellular location). |
| Strong staining appears in bone marrow hematopoietic cells, with little staining in the selected positive tissue. | Treat the contrast as unexpected: HPA reports low staining in bone marrow hematopoietic cells and high staining in the specified positive cell populations (HPA: tissue IHC). Review cell identification and controls for cross-reactivity or endogenous chromogenic activity. Low is not an HPA negative call (HPA: tissue IHC). |
| Color spreads across nuclei, cytoplasm, and tissue spaces without clear cell boundaries. | The diffuse pattern cannot support a nuclear localization call against the HPA reference pattern (HPA: ubiquitous nuclear expression). General IHC practice: inspect the no-primary control, wash conditions, blocking, and detection steps to distinguish background or endogenous activity from localized antibody staining. |
| No nuclear signal appears in an HPA high cell population. | A blank result conflicts with the reported high staining for that specific population (HPA: tissue IHC). Check that the expected cells are present, then review the antibody's IHC-P instructions, retrieval and detection controls as general IHC practice. HPA's Approved rating does not guarantee signal under every workflow (HPA: reliability description). |
💡Expected DNMT3A appearanceCall a result positive when nuclei predominate in an HPA high cell population with discernible staining; diffuse extracellular color or isolated nuclear-free staining is suspect (HPA: tissue IHC).
| Tissue and cell selection | Use a reported high cell population to judge whether staining can be detected; examples include bronchial respiratory epithelium, cerebellar granular-layer cells, colon endothelium, and breast adipocytes (HPA: tissue IHC). Bone marrow hematopoietic cells are reported low, not negative (HPA: tissue IHC). |
| Compartment and chromatin association | Nuclear staining is the principal IHC expectation (HPA: ubiquitous nuclear expression). DNMT3A also accumulates at pericentric heterochromatin (UniProt Q9Y6K1: subcellular location); the payload does not define a required punctate pattern on paraffin sections. |
| Antibody validation | HPA026588 and CAB009469 are each IHC Approved (HPA: antibody validation). The supplied antibody records do not label either IHC result Enhanced (HPA: antibody validation). HPA reports medium consistency between antibody staining and RNA expression, which limits how strongly an isolated staining pattern should be interpreted (HPA: reliability description). |
| Isoforms and epitope coverage | UniProt lists three DNMT3A isoforms and PWWP, ADD, and methyltransferase domains (UniProt Q9Y6K1: isoforms and domains). No antibody epitope is supplied, so the record cannot predict which isoforms a given antibody detects or attribute a missing stain to epitope placement. |
| IF/ICC Q&A: what localization is expected? | Predominantly nucleoplasmic signal is supported; vesicle localization is uncertain (HPA: subcellular ICC-IF). HPA026588 is ICC Supported, while no ICC status is supplied for CAB009469 (HPA: antibody validation). These ICC observations do not define a paraffin IHC protocol. |
| Situation | Likely cause | Next action |
|---|---|---|
| A reported high cell population is unstained. | The result disagrees with that population's high HPA staining level; the payload does not identify a DNMT3A-specific failure mechanism (HPA: tissue IHC). | Confirm the expected cells in the section. As general IHC practice, follow the catalog antibody's IHC-P instructions and check retrieval, primary antibody, detection reagents, and a positive control before calling the tissue negative. |
| Cytoplasmic color overwhelms nuclear color. | This is atypical for the HPA nuclear IHC profile, although UniProt lists cytoplasm among DNMT3A locations (HPA: tissue IHC; UniProt Q9Y6K1: subcellular location). | Compare the same run's positive tissue and no-primary control. As general IHC practice, inspect chromogen development and wash steps; retain a cytoplasmic interpretation only if localization and controls support it. |
| Staining is strong in bone marrow hematopoietic cells. | HPA reports low staining in these cells, so strong color warrants review; the report does not establish that they must be unstained (HPA: tissue IHC). | Verify cell identity and compare nuclear localization with a reported high population. As general IHC practice, use a no-primary control to assess endogenous detection activity before assigning specificity. |
| Diffuse color obscures nuclear boundaries. | The slide cannot be scored against the predominantly nuclear reference pattern (HPA: ubiquitous nuclear expression). Background sources cannot be distinguished from this appearance alone. | As general IHC practice, inspect a no-primary control, reagent blocking, washes, and chromogen development. Reassess nuclear localization only after cell boundaries and the counterstain are readable. |
| Only scattered cells stain in a reported high population. | The pattern is less widespread than the selected population's high HPA observation, but HPA does not prescribe a positive-cell percentage or score cutoff (HPA: tissue IHC). | Confirm section anatomy and compare similarly processed positive tissue. As general IHC practice, check section quality and detection steps before treating the scattered cells as a biological subgroup. |
| A result depends on a vesicle-like pattern or a single unexpected compartment. | HPA calls vesicle localization uncertain in ICC-IF, while its tissue IHC profile is nuclear (HPA: subcellular ICC-IF; tissue IHC). These observations do not validate vesicular paraffin-section staining. | Base the IHC call on identifiable nuclear staining in the expected cells and review controls. Record the unusual distribution separately; avoid assigning it to DNMT3A solely from its appearance. |
Experiment evidence
IHC & IF images showing DNMT3A staining
A00136-2 · IHCIHC analysis of DNMT3A using anti-DNMT3A antibody (A00136-2). DNMT3A was detected in a paraffin-embedded section of human colon cancer tissu
A00136-2 · IFIF analysis of DNMT3A using anti-DNMT3A antibody (A00136-2) and anti-Beta Tubulin antibody (M01857-3). DNMT3A was detected in an immunocytoc
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000119772-DNMT3A/tissue/cerebral+cortex#imid_6488432
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000119772-DNMT3A/tissue/colon#imid_6488358
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000119772-DNMT3A/tissue/liver#imid_6488359
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000119772-DNMT3A/tissue/kidney#imid_6488352
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA026588; green = DNMT3A, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA026588; green = DNMT3A, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for DNMT3A IHC & IF
🧪Run bronchus first: respiratory epithelial cells show High DNMT3A staining (HPA: High in bronchus respiratory epithelial cells). HPA detects DNMT3A in all 45 scored tissues, so there is no validated negative tissue; use no-primary and isotype controls, and treat cells without nuclear chromogen on the positive slide as background references rather than proven biological negatives (HPA: no negative tissue rows; UniProt Q9Y6K1: nucleus).
Positive control tissue: Adrenal gland (Glandular cells, HPA High)
Negative control tissue: None in HPA: DNMT3A 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 DNMT3A in A-431, U-251MG, U2OS, KOLF2.1J, with annotated localisation: Nucleoplasm (supported) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only slide, a concentration-matched rabbit IgG isotype control matched to the primary antibody’s clonality, and a matched DNMT3A-knockout specimen as a biological negative where available (caption: rabbit primary antibody; standard IHC controls). Block endogenous peroxidase for chromogenic detection and assess staining in inflammatory cells separately (caption: HRP/DAB detection; standard IHC practice).
⚠️Feasibility: No target-specific fixation window or fixation effect is reported in the supplied evidence; the A00136-2 paraffin-section caption does not state a fixative (caption: fixative unreported). That caption uses heat-mediated EDTA retrieval at pH 8.0, but does not establish whether retrieval is required for other specimens (caption: EDTA retrieval). The evidence does not establish that frozen sections or IF are easier; for bronchus, assess inflammatory-cell peroxidase background with the controls (HPA: High in bronchus respiratory epithelial cells; standard IHC practice).
HPA tissue IHC evidence for DNMT3A
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — 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 |
|---|---|---|---|---|
| Adrenal gland | Glandular cells | High | Protein (IHC) | HPA → |
| Appendix | Glandular cells | High | Protein (IHC) | HPA → |
| Breast | Adipocytes | High | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | High | Protein (IHC) | HPA → |
| Cerebellum | Cells in granular layer | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| None in HPA: DNMT3A is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead. | ||||
Section 3
Advanced DNMT3A IHC Tips
Troubleshoot DNMT3A staining in paraffin sections by checking retrieval, nuclear localisation, antibody specificity and cell-level scoring.
What retrieval should I try first when DNMT3A staining is weak?
Use heat-mediated antigen retrieval in EDTA at pH 8.0 first (datasheet A00136-2). The catalog antibody detected DNMT3A in a paraffin-embedded human colon cancer section after that retrieval, with 2 μg/ml primary antibody incubated overnight at 4°C (datasheet A00136-2). If nuclear staining remains weak, adjust heating time on adjacent sections while keeping buffer and antibody conditions constant, and assess tissue integrity alongside signal (standard IHC practice). Compare results in the same cell population because DNMT3A is predominantly nuclear, while uneven staining at section edges can reflect processing artefact (HPA tissue IHC; standard IHC practice).
Could fixation explain weak or uneven DNMT3A staining?
Target-specific fixation sensitivity is unknown from the supplied evidence; the catalog image identifies a paraffin-embedded section but does not report its fixative (datasheet A00136-2). Record the fixative, fixation duration and tissue thickness for each specimen, then compare sections processed together before changing antibody concentration (standard IHC practice). Check whether weak nuclei cluster in poorly preserved regions, since morphology and staining should be assessed together when diagnosing tissue processing problems (standard IHC practice). If fixation conditions differ between specimens, optimise retrieval on matched material and retain a common control section for each run; do not infer a DNMT3A-specific fixation effect from its localisation or modifications (standard IHC practice; UniProt Q9Y6K1).
How should I assess cytoplasmic DNMT3A staining in a tissue section?
Prioritise staining within intact nuclei: the tissue IHC profile describes ubiquitous nuclear expression, and nucleoplasm is the supported main subcellular location (HPA tissue IHC; HPA subcellular). UniProt also lists chromosome and cytoplasm and describes accumulation at pericentric heterochromatin, so some nonuniform nuclear staining is biologically plausible (UniProt Q9Y6K1). Score nuclear and cytoplasmic signals separately rather than merging them into one positive category, and compare each with the matched control and tissue morphology (standard IHC practice). Predominantly diffuse cytoplasmic DAB without convincing nuclear staining warrants checking retrieval, antibody background and detection controls before assigning it to DNMT3A (HPA tissue IHC; standard IHC practice).
Can epitope placement change which DNMT3A forms I detect?
DNMT3A has 3 listed isoforms, but the supplied catalog image does not identify the antibody epitope or establish isoform-specific IHC detection (UniProt Q9Y6K1; datasheet A00136-2). Map the immunogen sequence against each isoform before interpreting a negative cell population as lacking all DNMT3A forms (standard IHC practice; UniProt Q9Y6K1). The annotated PWWP, ADD and catalytic domains span residues 292–350, 482–614 and 634–912, respectively; modifications include phosphorylation at residues 105 and 243 (UniProt Q9Y6K1). Without epitope mapping, neither domain position nor modification status establishes whether this antibody distinguishes isoforms or modification states in paraffin sections (standard IHC practice; UniProt Q9Y6K1).
How can I check a nuclear DNMT3A pattern by multiplex IF?
Choose a marker for the cell population being examined and include a nuclear counterstain, then assess DNMT3A signal within those identified nuclei (HPA tissue IHC; standard IF practice). For example, HPA reports high staining in colon endothelial cells; use a validated endothelial marker if that population is the question (HPA tissue IHC; standard IF practice). Place the weaker signal in a fluorophore channel with low tissue autofluorescence and inspect unstained or single-stained controls for bleed-through (standard IF practice). Because DNMT3A is mainly nucleoplasmic and has no transmembrane segment, optimise permeabilisation for access to its nuclear epitope after fixation; the catalog paraffin IHC caption supplies no IF fixation or permeabilisation conditions (HPA subcellular; UniProt Q9Y6K1; datasheet A00136-2).
What should I check when DAB obscures DNMT3A-positive nuclei?
Inspect a section without primary antibody to separate detection-system deposits from primary-antibody-associated staining, and review the peroxidase block before DAB development (standard IHC practice). The catalog image used a goat-serum block of 10%, a peroxidase-conjugated secondary incubated for 30 minutes at 37°C, and DAB detection (datasheet A00136-2). If diffuse staining persists, titrate primary antibody around the documented 2 μg/ml condition and shorten chromogen development while comparing nuclear contrast on adjacent sections (datasheet A00136-2; standard IHC practice). Judge apparent positives against intact cell morphology and the expected nuclear distribution, especially near folds, damaged regions and section edges (HPA tissue IHC; standard IHC practice).
How should I quantify DNMT3A across specimens with different cellularity? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and analysis area before scoring, and count only interpretable nuclei within intact tissue (standard IHC practice). Report the percentage of DNMT3A-positive nuclei and, when intensity is reproducible across a common staining run, a nuclear H-score using 0–3 intensity categories (standard IHC practice). Normalise positive counts to the number of eligible nuclei in that population, or report positive-cell density per mm² of analysed tissue when area is the relevant denominator (standard IHC practice). Keep staining batches, thresholds and excluded regions documented because the HPA tissue profile is broadly nuclear but has only medium consistency between staining and RNA expression data (HPA tissue IHC; standard IHC practice).
When is DNMT3A staining convincing rather than an artefact?
Convincing staining identifies intact nuclei in the expected cell population and remains interpretable beside a matched control; HPA describes broadly nuclear tissue staining (HPA tissue IHC; standard IHC practice). High HPA signals include colon endothelial cells and appendix glandular cells, while bone-marrow hematopoietic cells are listed as low, so record which cells were actually scored (HPA tissue IHC). Treat signal concentrated at section edges, folds or necrotic areas cautiously, and use a no-primary control to assess endogenous enzyme or detection background (standard IHC practice). A predominantly cytoplasmic pattern needs independent validation because the supported HPA main location is nucleoplasm, although UniProt also lists cytoplasm (HPA subcellular; UniProt Q9Y6K1).
Boster reagents
Best DNMT3A / DNA (cytosine-5)-methyltransferase 3A IHC Antibodies
A00136-2 has real IHC data in human colon cancer paraffin sections and IF data in HeLa cells (catalog image captions). Both antibodies list human, mouse and rat reactivity (catalog reactivity).
Real IHC data
Anti-DNMT3A Antibody ®
Cat # A00136-2
A00136-2 is shown in human colon cancer paraffin sections by IHC and in HeLa cells by IF (A00136-2 image captions). M00136 lists IHC and IF/ICC applications and human, mouse and rat reactivity, but has no supplied IHC or IF figure (M00136 catalog entry).
Which to pick: Choose A00136-2 for tissue IHC: its image shows human colon cancer paraffin sections with EDTA heat retrieval and 2 μg/ml primary antibody; the fixative is unreported (A00136-2 IHC image caption). Choose A00136-2 for IF/ICC when a documented cell image matters: its HeLa IF image used 5 μg/ml primary antibody (A00136-2 IF image caption). For mouse or rat samples, both SKUs list reactivity, but neither supplied image demonstrates those species; M00136 is a rabbit monoclonal with IHC and IF/ICC listed if clonality is the deciding factor (catalog reactivity, applications and clone fields; image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q9Y6K1 (DNM3A_HUMAN, DNA (cytosine-5)-methyltransferase 3A).
- Human Protein Atlas. DNMT3A tissue IHC expression (reliability: Approved).
- Human Protein Atlas. DNMT3A subcellular location (ICC-IF): Mainly localized to the nucleoplasm. In addition localized to vesicles..
- Human Protein Atlas. DNMT3A antibody validation summary (2 antibodies).
- A functional polymorphism in the DNA methyltransferase-3A promoter modifies the susceptibility in gastric cancer but not in esophageal carcinoma. BMC medicine 2010 — PMC2829483.
- Sohlh2 Inhibits the Malignant Progression of Renal Cell Carcinoma by Upregulating Klotho via DNMT3a. Frontiers in oncology 2021 — PMC8807643.
- Expression of DNMTs and H3K9ac in Ameloblastoma and Ameloblastic Carcinoma. Frontiers in oral health 2021 — PMC8757744.
- An epigenetic regulator-related score (EpiScore) predicts survival in patients with diffuse large B cell lymphoma and identifies patients who may benefit from epigenetic therapy. Oncotarget 2018 — PMC5922379.
- PubMed PMID:10433969 — UniProt-cited evidence.
- PubMed PMID:12138111 — UniProt-cited evidence.
- PubMed PMID:12145218 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for DNMT3A (DNA (cytosine-5)-methyltransferase 3A). Expected localisation Nuclear staining in tissue (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A00136-2). Validated IHC antibody: A00136-2 at 2-5 μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Adrenal gland (Glandular cells); negative None in HPA (detected in all 45 tissues); use no-primary + isotype controls. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human colon cancer tissue; fixative not specified (datasheet A00136-2); 2. Fixation: Image fixative and duration unreported (datasheet A00136-2); 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 A00136-2); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A00136-2); 8. Primary antibody: Rabbit anti-DNMT3A, 2-5 μg/ml (datasheet A00136-2); 9. Primary incubation: Overnight at 4 °C (datasheet A00136-2). Watch for: Fixation: Keep fixation conditions consistent across samples (standard IHC practice; not target-specific); Caveat: Bone marrow hematopoietic cells show low staining (HPA tissue IHC); Isoform / epitope: 3 isoforms; verify epitope coverage (UniProt); Regulation: Higher expression in fetal tissues (UniProt). Evidence: UniProt Q9Y6K1, HPA tissue IHC · Approved, HPA ICC-IF · Mainly localized to the nucleoplasm. In addition localized to…, Datasheet A00136-2, PMC2829483, PMC8757744. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/DNMT3A/immunohistochemistry-ihc-guide.