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ASH2L / Set1/Ash2 histone methyltransferase complex subunit ASH2 · IHC design guide
Design Immunohistochemistry for ASH2L
Plan chromogenic ASH2L IHC in paraffin sections around mainly nuclear staining (HPA tissue IHC). Use placenta decidual cells as a medium-staining reference (HPA tissue IHC) and start the catalog antibody at 2–5 μg/mL (datasheet A06129-1).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
ASH2L 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 | Mainly nuclear staining across several tissues (HPA tissue IHC) | |
| Staining pattern | Nuclear staining in glandular, tubular and squamous cells (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A06129-1) | |
| Positive control | Appendix+4 more · see all | |
| Negative control | Caudate+4 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent across sections (standard IHC practice; not target-specific) | |
| Caveat | IHC staining has low consistency with RNA expression (HPA tissue IHC) | |
| Regulation | Stimulus-linked regulation not specified (UniProt) | |
| Isoform / epitope | 3 isoforms; epitope coverage is unspecified (UniProt) |
Section 1
Recommended ASH2L IHC & IF Protocols
Compare the catalog antibody’s IHC-P protocol (datasheet: A06129-1) with one published ASH2L testis protocol (PMC10215499).
| Sample | Paraffin-embedded human lung adenocarcinoma tissue; fixative not specified (datasheet A06129-1) |
| Fixation | Image fixative and duration unreported (datasheet A06129-1); 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 A06129-1); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A06129-1) |
| Primary antibody | Rabbit anti-ASH2L, 2-5 μg/ml (datasheet A06129-1) |
| Primary incubation | Overnight at 4 °C (datasheet A06129-1) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet A06129-1) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | ASH2L-positive staining in glandular cells of appendix (HPA tissue IHC: Medium). HPA tissue profile: Mainly nuclear expression in several different tissue types. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA retrieval at pH 8.0 for the catalog antibody (datasheet: A06129-1); the published excerpt does not specify retrieval (PMC10215499).
Section 2
What Is the Expected ASH2L Staining Pattern?
ASH2L is a nuclear protein without a transmembrane segment (UniProt Q9UBL3). In paraffin tissue sections, expect mainly nuclear staining in several cell types, including glandular cells and selected epithelial cells (HPA tissue IHC). HPA rates its tissue IHC profile Approved but reports low consistency between antibody staining and RNA expression; interpret individual tissue signals with that limitation in mind (HPA tissue IHC).
| Distinct nuclear staining in appendix or colon glandular cells, or esophageal squamous epithelial cells. | This fits the reported medium staining in those specific cell populations and the mainly nuclear tissue profile (HPA tissue IHC). Assess the named cells within each section; an entire organ should not be scored uniformly positive from these entries alone (HPA tissue IHC). |
| Predominantly diffuse cytoplasmic staining with little or no nuclear signal. | That distribution is inconsistent with the expected tissue IHC pattern and nuclear localization (HPA tissue IHC; UniProt Q9UBL3). Review background and detection controls before calling it ASH2L. HPA also reports plasma membrane localization in ICC-IF, so a membrane signal in that separate application needs its own assessment (HPA subcellular ICC-IF). |
| Strong staining in a cell population HPA lists as not detected, such as lung alveolar cells. | The result conflicts with that cell-specific HPA observation and merits scrutiny for cross-reactivity or endogenous detection activity (HPA tissue IHC; standard IHC practice). An HPA 'not detected' entry describes the sampled cell population; it does not establish that every cell in the tissue is negative (HPA tissue IHC). |
| Diffuse color across tissue, lumens, and spaces between cells, with no clear nuclear boundaries. | This is background rather than a convincing mainly nuclear pattern (HPA tissue IHC; standard IHC practice). Check the no-primary control, wash steps, blocking, and chromogen development as general IHC troubleshooting steps (standard IHC practice). Background alone cannot establish ASH2L expression (standard IHC practice). |
| No nuclear signal in appendix or colon glandular cells on an otherwise interpretable section. | Those cells showed medium staining in the HPA tissue profile, so a blank result warrants a technical check (HPA tissue IHC). Compare section quality and positive-control staining, then review the catalog antibody's IHC-P conditions (standard IHC practice). HPA's Approved rating and low RNA–staining consistency limit how strongly one negative section can be interpreted (HPA tissue IHC). |
💡Expected ASH2L appearanceCall a positive result when defined nuclei stain in HPA-listed positive cell populations at a level consistent with the reported medium examples; diffuse nonnuclear color alone is unconvincing (HPA tissue IHC; standard IHC practice).
| Compartment and application | UniProt places ASH2L in the nucleus; HPA tissue IHC describes mainly nuclear expression (UniProt Q9UBL3; HPA tissue IHC). HPA ICC-IF additionally supports plasma membrane localization, which should be interpreted within ICC-IF rather than used to redefine the tissue IHC pattern (HPA subcellular ICC-IF). |
| Cell-specific tissue distribution | HPA reports medium staining in kidney tubule cells, ovary follicle cells, and placenta decidual cells, while lung alveolar cells and liver cholangiocytes were not detected (HPA tissue IHC). Select and score the documented cell population, since these entries do not describe every cell in each organ (HPA tissue IHC). |
| Validation and RNA agreement | The listed antibody HPA042289 is Approved for IHC and ICC; the tissue profile is Approved with low consistency between staining and RNA expression (HPA antibodies; HPA tissue IHC). Treat RNA abundance as context, not a substitute for cell-specific staining assessment in a section (HPA tissue IHC). |
| Isoforms and epitope information | UniProt lists 3 ASH2L isoforms, but this payload gives no antibody epitope or isoform coverage (UniProt Q9UBL3; supplied HPA antibody record). An isoform-specific explanation for an absent signal would therefore be unverified; check the catalog antibody documentation before making that interpretation. |
| Protein architecture and processing | ASH2L has no transmembrane segment, signal peptide, or propeptide; UniProt lists a chain spanning residues 1–628 (UniProt Q9UBL3). These facts support a nuclear expectation but do not establish antigen-retrieval conditions or target-specific sensitivity to fixation (UniProt Q9UBL3). |
| Situation | Likely cause | Next action |
|---|---|---|
| Expected nuclei are blank in a positive-control appendix or colon section. | The assay may have failed, or the sampled glandular cells may be difficult to evaluate; HPA reports medium staining in those cells (HPA tissue IHC; standard IHC practice). | Verify tissue identity and morphology, then check the catalog antibody's IHC-P dilution and retrieval instructions and the detection run (standard IHC practice). Do not infer a target-specific fixation effect from this result alone. |
| Color appears mainly in cytoplasm rather than nuclei. | The compartment conflicts with UniProt nuclear localization and HPA's mainly nuclear tissue IHC profile; nonspecific staining is possible (UniProt Q9UBL3; HPA tissue IHC; standard IHC practice). | Compare with a no-primary control and a documented positive cell population; review blocking, washes, and detection conditions before scoring it as specific (standard IHC practice). |
| A cell population listed as not detected stains strongly. | Cross-reactivity or endogenous detection activity is possible, although HPA's entry applies only to its named cell population and observations (HPA tissue IHC; standard IHC practice). | Confirm the cell identity and compartment, inspect the no-primary control, and compare a documented positive population in the same run (HPA tissue IHC; standard IHC practice). |
| Brown chromogen is widespread with weak nuclear contrast. | Background or excess chromogen development can obscure a nuclear pattern (standard IHC practice). | Review blocking, washing, antibody dilution, and development time against the established IHC-P workflow; judge signal against nuclear boundaries and the control section (standard IHC practice). |
| A low-staining tissue is being used to declare antibody failure. | HPA reports low staining in some named populations, including duodenal glandular cells and bronchial respiratory epithelial cells (HPA tissue IHC). A faint result there offers a limited performance check. | Use a documented medium-staining cell population, such as appendix glandular cells, as the positive comparator and score the specified cells separately (HPA tissue IHC; standard IHC practice). |
| IF/ICC shows membrane staining alongside nucleoplasmic signal. | HPA supports nucleoplasm as the main ICC-IF location and plasma membrane as an additional location (HPA subcellular ICC-IF). That observation comes from ICC-IF rather than paraffin tissue IHC. | Assess the ICC-IF result within its own guide and controls; for this IHC section, use HPA's mainly nuclear tissue pattern to interpret chromogenic staining (HPA subcellular ICC-IF; HPA tissue IHC). |
Experiment evidence
IHC & IF images showing ASH2L staining
A06129-1 · IHCIHC analysis of ASH2L using anti-ASH2L antibody (A06129-1). ASH2L was detected in a paraffin-embedded section of human lung adenocarcinoma t
A06129-1 · IHCIHC analysis of ASH2L using anti-ASH2L antibody (A06129-1). ASH2L was detected in a paraffin-embedded section of human lung adenocarcinoma t
A06129-1 · IFIF analysis of ASH2L using anti-ASH2L antibody (A06129-1) and anti-Beta Tubulin antibody (M01857-3). ASH2L was detected in immunocytochemica
M06129 · IHCImmunohistochemical analysis of paraffin-embedded human kideny, using ASH2L Antibody.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000129691-ASH2L/tissue/cerebral+cortex#imid_11472559
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000129691-ASH2L/tissue/colon#imid_11472493
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000129691-ASH2L/tissue/liver#imid_11472486
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000129691-ASH2L/tissue/kidney#imid_11472495
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA042289; green = ASH2L, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA042289; green = ASH2L, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for ASH2L IHC & IF
🧪Run appendix first and look for ASH2L staining in glandular cells (Medium; HPA: appendix glandular cells). Use caudate glial cells as the negative tissue (Not detected; HPA: caudate glial cells); on the appendix slide, cells without nuclear DAB staining should retain visible counterstained nuclei, but HPA does not identify a validated internal negative cell type (HPA: appendix glandular cells).
Positive control tissue: Appendix (Glandular cells, HPA Medium)
Negative control tissue: Caudate (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show ASH2L in A-431, U-251MG, U2OS, with annotated localisation: Nucleoplasm (supported) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control, a matched nonimmune rabbit IgG control for the caption’s rabbit primary, and an ASH2L knockout control where available (selected-SKU IHC caption: rabbit primary; standard IHC practice). Quench endogenous peroxidase before chromogenic detection and check inflammatory cells in appendix for background staining (standard IHC practice).
⚠️Feasibility: No ASH2L-specific fixation window or fixation effect is reported, and the selected-SKU paraffin-section caption does not state a fixative (selected-SKU IHC caption: fixative unreported). The caption uses heat-mediated retrieval in EDTA at pH 8.0; it does not establish that retrieval is required (selected-SKU IHC caption). The supplied evidence does not establish that frozen sections or IF are easier than paraffin IHC; assess appendix inflammatory cells for peroxidase-related background when interpreting DAB staining (HPA: appendix; standard IHC practice).
HPA tissue IHC evidence for ASH2L
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Low 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 |
|---|---|---|---|---|
| Appendix | Glandular cells | Medium | Protein (IHC) | HPA → |
| Colon | Glandular cells | Medium | Protein (IHC) | HPA → |
| Esophagus | Squamous epithelial cells | Medium | Protein (IHC) | HPA → |
| Fallopian tube | Glandular cells | Medium | Protein (IHC) | HPA → |
| Gallbladder | Glandular cells | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Caudate | Glial cells | Not detected | Protein (IHC) | HPA → |
| Cerebellum | Cells in granular layer | Not detected | Protein (IHC) | HPA → |
| Hippocampus | Glial cells | Not detected | Protein (IHC) | HPA → |
| Liver | Cholangiocytes | Not detected | Protein (IHC) | HPA → |
| Lung | Alveolar cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced ASH2L IHC Tips
Use the IHC-validated antibody’s paraffin-section workflow as a starting point, then assess staining by cell type and compartment (datasheet A06129-1; HPA tissue IHC).
How should I troubleshoot weak ASH2L staining after antigen retrieval?
Start with heat-mediated antigen retrieval in EDTA at pH 8.0 for paraffin sections (datasheet A06129-1). The tested image used 2 μg/ml primary antibody overnight at 4°C, so first check that those conditions and the retrieval solution were reproduced (datasheet A06129-1). If nuclear staining remains weak, compare retrieval heating times on adjacent sections while keeping antibody and detection conditions constant (standard IHC practice; UniProt Q9UBL3 localisation). Include a known staining reference, such as appendix glandular cells, in each comparison (HPA tissue IHC: Medium). Excessive heating can damage tissue morphology and make nuclear scoring unreliable (standard IHC practice).
Could fixation explain weak or uneven ASH2L staining in paraffin sections?
The selected paraffin-section caption does not state a fixative, so ASH2L-specific sensitivity to fixation is unknown (datasheet A06129-1). Record the fixative and processing history for each specimen before comparing staining intensity across cases (standard IHC practice). Use adjacent sections from the same block to assess changes in retrieval, keeping EDTA at pH 8.0 and other conditions matched (datasheet A06129-1; standard IHC practice). Inspect tissue preservation and section adhesion alongside nuclear staining, since damaged sections can distort an intensity comparison (standard IHC practice). Do not infer fixation performance from the reported tissue staining pattern or protein modifications (HPA tissue IHC; UniProt Q9UBL3).
How should I assess ASH2L staining outside the nucleus?
Prioritise nuclear staining when assessing the chromogenic section: ASH2L is annotated as nuclear, and tissue IHC shows mainly nuclear expression (UniProt Q9UBL3 localisation; HPA tissue IHC). Supported nucleoplasmic and additional plasma-membrane locations have been reported in subcellular imaging, so document distinct patterns separately (HPA subcellular). ASH2L has no annotated transmembrane segment, making a membrane-only DAB pattern worth checking against controls and morphology (UniProt Q9UBL3 topology; standard IHC practice). Compare cellular borders with the counterstained nuclei at the same focal plane before calling membrane signal (standard IHC practice). Score nuclear and extranuclear staining separately rather than combining them into one intensity value (standard IHC practice).
Could isoforms or epitope accessibility explain discordant ASH2L staining?
ASH2L has 3 annotated isoforms, but the supplied antibody caption does not identify its epitope or establish isoform selectivity (UniProt Q9UBL3 isoforms; datasheet A06129-1). Its B30.2/SPRY domain spans residues 360–583, and annotated modifications include serines 101 and 316 and arginine 296 (UniProt Q9UBL3). Those annotations alone cannot show whether processing masks this antibody’s epitope or whether a given isoform is detected (UniProt Q9UBL3; standard IHC interpretation). Check the antibody’s documented immunogen before assigning a negative section to isoform absence (standard IHC practice). If available, compare an independently validated antibody against a known, different epitope on matched sections (standard IHC practice).
How can IF help clarify an ambiguous chromogenic ASH2L pattern?
Use IF as a separate localisation check while retaining the paraffin chromogenic result as the IHC readout (standard IHC/IF practice). Multiplex ASH2L with a marker for the expected cell population, such as a tubular epithelial marker when examining kidney tubules, which show medium IHC staining (HPA tissue IHC). Choose fluorophores away from prominent tissue autofluorescence and include single-label controls to assess bleed-through (standard IF practice). For a nuclear epitope, use permeabilisation that permits antibody access to nuclei; the specific epitope side is unreported for this antibody (UniProt Q9UBL3 localisation; datasheet A06129-1). Compare nuclear signal with counterstained nuclei and record any separate membrane pattern (HPA subcellular; standard IF practice).
What should I check when ASH2L DAB staining looks diffuse?
First compare the specimen with a no-primary control to identify signal from the detection system or endogenous tissue activity (standard IHC practice). The documented paraffin-section workflow used 10% goat serum blocking, 2 μg/ml primary antibody overnight at 4°C, and HRP/DAB detection (datasheet A06129-1). Apply an appropriate endogenous peroxidase block before HRP detection and check whether DAB develops in the no-primary control (standard IHC practice). Review wash consistency, section edges, pigment, and damaged areas before interpreting diffuse colour as cellular staining (standard IHC practice). Expect predominantly nuclear tissue staining and score diffuse extranuclear colour separately (HPA tissue IHC; standard IHC practice).
How should I quantify ASH2L across heterogeneous tissue regions? ⚠ ANSWER MARKED FOR VERIFICATION
Define the target cell population and viable regions before scoring, then assess nuclear DAB within those cells (UniProt Q9UBL3 localisation; standard IHC practice). Report percent positive nuclei and intensity categories 0–3; an H-score from 0–300 combines both measures (standard IHC practice). Alternatively, report positive nuclei per mm² when cell density itself is the outcome (standard IHC practice). Normalise intensity measures to the viable, nucleated target cells examined, and state the denominator for density measurements (standard IHC practice). Keep cell types separate, since the tissue atlas reports medium staining in kidney tubules but no detection in lung alveolar cells (HPA tissue IHC).
How can I distinguish genuine ASH2L staining from artefact?
A convincing result has reproducible staining in identifiable cells, with a nuclear component consistent with ASH2L’s annotated location and tissue IHC pattern (UniProt Q9UBL3 localisation; HPA tissue IHC). Compare cell identity carefully: the selected lung adenocarcinoma section shows staining, whereas lung alveolar cells were not detected in the atlas (datasheet A06129-1; HPA tissue IHC). Treat staining limited to section edges, necrotic areas, or a no-primary control as suspect (standard IHC practice). Check endogenous peroxidase activity before attributing DAB deposits to ASH2L (standard IHC practice). Interpret apparent membrane signal separately, because subcellular imaging also reports a supported plasma-membrane location and atlas IHC has low RNA concordance (HPA subcellular; HPA tissue IHC).
Boster reagents
Best ASH2L / Set1/Ash2 histone methyltransferase complex subunit ASH2 IHC Antibodies
Two anti-ASH2L antibodies list human, mouse and rat reactivity and have human paraffin-section IHC images; A06129-1 also has a HELA-cell IF image (catalog reactivity; image captions).
Real IHC data
Anti-ASH2L Antibody ®
Cat # A06129-1
Real IHC data
Anti-ASH2L Rabbit Monoclonal Antibody
Cat # M06129
A06129-1 has IHC images from paraffin-embedded human lung adenocarcinoma and placenta, plus an IF image from HELA cells (catalog image captions). M06129 has a paraffin-embedded human kidney IHC image and lists IHC and IF/ICC applications (catalog IHC caption; catalog applications).
Which to pick: For tissue IHC, A06129-1 provides an EDTA pH 8.0 retrieval and DAB example; M06129 is a rabbit monoclonal with a human kidney paraffin-section image (A06129-1 IHC caption; M06129 catalog title and IHC caption). For IF/ICC, A06129-1 has a HELA-cell IF image, while M06129 lists IF/ICC without an IF image (catalog IF images; catalog applications). Both list human, mouse and rat reactivity, but their supplied images show human samples only; neither IHC caption reports the fixative (catalog reactivity; image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q9UBL3 (ASH2L_HUMAN, Set1/Ash2 histone methyltransferase complex subunit ASH2).
- Human Protein Atlas. ASH2L tissue IHC expression (reliability: Approved).
- Human Protein Atlas. ASH2L subcellular location (ICC-IF): Mainly localized to the nucleoplasm. In addition localized to the plasma membrane..
- Human Protein Atlas. ASH2L antibody validation summary (1 antibodies).
- ASH2L is involved in promotion of endometrial cancer progression via upregulation of PAX2 transcription. Cancer science 2020 — PMC7293091.
- Induction of Hibernation and Changes in Physiological and Metabolic Indices in Pelodiscus sinensis. Biology 2023 — PMC10215499.
- Ash2l deficiency impairs adipose tissue thermogenesis and exacerbates obesity in mice. Cellular & molecular biology letters 2026 — PMC13045091.
- PubMed PMID:11466562 — UniProt-cited evidence.
- PubMed PMID:10393421 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for ASH2L (Set1/Ash2 histone methyltransferase complex subunit ASH2). Expected localisation Mainly nuclear staining across several tissues (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A06129-1). Validated IHC antibody: A06129-1 at 2-5 μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Appendix (Glandular cells); negative Caudate. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human lung adenocarcinoma tissue; fixative not specified (datasheet A06129-1); 2. Fixation: Image fixative and duration unreported (datasheet A06129-1); 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 A06129-1); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A06129-1); 8. Primary antibody: Rabbit anti-ASH2L, 2-5 μg/ml (datasheet A06129-1); 9. Primary incubation: Overnight at 4 °C (datasheet A06129-1). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific); Caveat: IHC staining has low consistency with RNA expression (HPA tissue IHC); Isoform / epitope: 3 isoforms; epitope coverage is unspecified (UniProt); Regulation: Stimulus-linked regulation not specified (UniProt). Evidence: UniProt Q9UBL3, HPA tissue IHC · Approved, HPA ICC-IF · Mainly localized to the nucleoplasm. In addition localized to the…, Datasheet A06129-1, PMC10215499. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/ASH2L/immunohistochemistry-ihc-guide.