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ASH2L / Set1/Ash2 histone methyltransferase complex subunit ASH2 · IHC design guide
Design Immunohistochemistry for ASH2L
Plan ASH2L chromogenic IHC on paraffin sections using the mainly nuclear tissue pattern (HPA tissue IHC). This guide covers the catalog antibody’s documented IHC workflow (datasheet A06129-1) and the staining-to-RNA discordance to consider when interpreting results (HPA tissue IHC).
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 in several tissues (HPA tissue IHC) | |
| Staining pattern | Nuclear staining in several tissue cell types (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 paraffin-section fixation consistent (standard IHC practice; not target-specific); Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A06129-1) | |
| Caveat | Staining has low concordance with RNA expression (HPA tissue IHC) | |
| Regulation | Higher expression in adult heart and testis (UniProt) | |
| Isoform / epitope | 3 isoforms; epitope differences unspecified (UniProt) |
Section 1
Recommended ASH2L IHC & IF Protocols
The catalog antibody has an IHC-P protocol (datasheet A06129-1); one published ASH2L protocol reports staining of Pelodiscus sinensis testis (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).
Section 2
What Is the Expected ASH2L Staining Pattern?
ASH2L is a nuclear protein with no transmembrane segment (UniProt Q9UBL3). In paraffin tissue sections, expect mainly nuclear staining in several cell types, including appendix and colon glandular cells and kidney tubule cells with medium reported staining (HPA tissue IHC). HPA rates its IHC staining Approved but reports low consistency with RNA expression data, so use cell-specific tissue patterns as guides rather than universal positive controls (HPA tissue IHC).
| Medium nuclear staining in appendix or colon glandular cells, or kidney tubule cells (HPA tissue IHC). | This matches the reported cell-specific IHC pattern and the nuclear localisation of ASH2L (HPA tissue IHC; UniProt Q9UBL3). Compare nuclei with nearby background before scoring, and record the stained cell type as well as intensity (general IHC practice). |
| Predominantly cytoplasmic staining, with little nuclear signal, in an otherwise positive field. | This does not match the mainly nuclear tissue pattern (HPA tissue IHC). Check morphology, chromogen deposits and control sections before interpreting it as ASH2L; ICC-IF also reports a supported plasma membrane location, but that observation alone does not establish a matching paraffin IHC pattern (HPA subcellular; general IHC practice). |
| Staining in a cell population reported as undetected, such as lung alveolar cells (HPA tissue IHC). | Treat this as unexpected for that specific population, not proof that the entire lung must be negative (HPA tissue IHC). Cross-reactivity or endogenous detection activity is possible; review an antibody-omission control and cell identity before scoring (general IHC practice). |
| Diffuse colour across nuclei, cytoplasm and acellular areas. | A diffuse field cannot establish nuclear localisation (UniProt Q9UBL3; general IHC practice). Background from detection reagents, incomplete blocking or inadequate washing is possible; compare an antibody-omission control and inspect whether the signal follows tissue structures (general IHC practice). |
| No nuclear signal in appendix or colon glandular cells (HPA tissue IHC). | These cells were reported at medium staining, so an absent signal warrants a run-level check (HPA tissue IHC). The HPA Approved rating has low staining-to-RNA consistency; a single negative section cannot establish absence of ASH2L protein (HPA tissue IHC). |
💡Expected ASH2L appearanceCall a result positive when the expected cells show predominantly nuclear, roughly medium-intensity staining, as reported for appendix or colon glandular cells and kidney tubule cells; broad cytoplasmic colour or staining confined to cell populations reported as undetected needs control review (HPA tissue IHC; UniProt Q9UBL3; general IHC practice).
| Compartment and topology (UniProt Q9UBL3). | ASH2L is nuclear and has no transmembrane segment, making nuclear staining the main IHC readout (UniProt Q9UBL3; HPA tissue IHC). Do not infer that every membrane-like signal is false: supported plasma membrane localisation was observed by ICC-IF (HPA subcellular). |
| Cell-specific reference patterns and validation (HPA tissue IHC). | Several epithelial and other populations show medium staining, while named populations such as lung alveolar cells and liver cholangiocytes were not detected (HPA tissue IHC). The Approved IHC assessment also notes low consistency with RNA data; interpret controls by cell type (HPA tissue IHC). |
| Three reported isoforms (UniProt Q9UBL3). | Isoforms 1, 2 and 3 are listed, but the supplied record gives no antibody epitope or isoform-specific tissue pattern (UniProt Q9UBL3). A positive or negative IHC result cannot be assigned to one isoform from these data alone (UniProt Q9UBL3). |
| Unreported target-specific preparation response. | Target-specific fixation effects are not established by the supplied assay evidence. Verify with a matched IHC source before attributing a result to fixation. |
| Situation | Likely cause | Next action |
|---|---|---|
| No signal appears in expected appendix or colon glandular cells (HPA tissue IHC). | Possible run failure, inadequate detection, or a section-dependent result; HPA reports medium staining in these cells but low consistency with RNA data (HPA tissue IHC; general IHC practice). | Confirm the expected cell type and morphology, inspect a known-working positive section processed in the same run, then review reagent sequence and detection steps (HPA tissue IHC; general IHC practice). |
| Signal is mainly cytoplasmic instead of nuclear. | The compartment differs from the predominant tissue IHC pattern and UniProt localisation (HPA tissue IHC; UniProt Q9UBL3). Nonspecific staining or deposited chromogen may account for it (general IHC practice). | Compare antibody-omission and positive controls, inspect deposits at higher magnification, and score nuclear signal separately from cytoplasmic colour (general IHC practice). |
| Cells reported as undetected show convincing-looking colour, such as liver cholangiocytes (HPA tissue IHC). | Cross-reactivity, endogenous detection activity, or mistaken cell identification is possible (general IHC practice). The HPA observation refers to cholangiocytes, not every cell in liver (HPA tissue IHC). | Check cell morphology and an antibody-omission control; if colour persists without primary antibody, investigate the detection system before assigning ASH2L positivity (general IHC practice). |
| Background obscures nuclear boundaries throughout the section. | Nonspecific reagent binding, endogenous detection activity, or incomplete washing can reduce contrast (general IHC practice). The diffuse pattern does not resolve the expected nuclear signal (HPA tissue IHC). | Review blocking, washing and detection controls using standard IHC practice; score only cells whose nuclei can be distinguished reliably (general IHC practice). |
| Intensity differs across tissues or between nearby cell populations. | HPA reports medium staining in selected populations, low staining in others, and no detection in some named populations; its antibody assessment notes low consistency with RNA data (HPA tissue IHC). | Record tissue, cell type, compartment and intensity separately. Compare each population with its own HPA observation instead of applying a whole-tissue positive or negative label (HPA tissue IHC; general IHC practice). |
| Does an ICC-IF membrane signal change the expected paraffin IHC result? | HPA ICC-IF lists nucleoplasm as the main location and plasma membrane as an additional supported location (HPA subcellular). Tissue IHC is described as mainly nuclear (HPA tissue IHC). | Use predominantly nuclear staining for the IHC decision; assess ICC-IF localisation on its own guide page, and do not infer a paraffin IHC membrane pattern from ICC-IF alone (HPA tissue IHC; HPA subcellular). |
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: its glandular cells show medium ASH2L staining (HPA: Appendix, glandular cells, Medium). Use caudate glial cells as the negative comparator (HPA: Caudate, glial cells, Not detected); on the appendix slide, treat cells lacking nuclear signal as internal background references, without assuming a particular cell type is ASH2L-negative (UniProt Q9UBL3: ubiquitous expression; nucleus).
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 slide and an isotype-matched nonimmune rabbit IgG control (selected-SKU caption: rabbit primary; standard IHC control practice). Confirm specificity with ASH2L-knockout material if available, and block endogenous peroxidase before HRP/DAB detection in appendix sections (standard IHC control practice; selected-SKU caption: HRP/DAB).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected-SKU tissue-IHC caption does not state the fixative (selected-SKU caption: fixative not stated). The caption demonstrates heat retrieval in EDTA at pH 8.0 for a paraffin section, but does not establish whether retrieval is required under other conditions or whether frozen sections are easier (selected-SKU caption: EDTA retrieval, paraffin section). IF/ICC images exist for A-431, U-251MG and U2OS, with mainly nucleoplasmic signal and additional plasma-membrane signal; the supplied evidence does not establish that IF is easier than tissue IHC (HPA subcellular: ICC-IF images and supported locations).
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
Troubleshoot ASH2L staining in paraffin sections by checking retrieval, nuclear localisation, controls, and cell specific scoring before interpreting differences between tissues.
Which retrieval condition should I start with for ASH2L in paraffin sections?
Start with heat mediated retrieval in EDTA at pH 8.0 for paraffin sections (datasheet A06129-1). The selected tissue IHC caption used this retrieval with 2 μg/ml primary antibody overnight at 4°C; keep those conditions fixed while comparing heating duration on serial sections (datasheet A06129-1; standard IHC practice). If nuclear staining remains weak, inspect morphology and section adhesion before trying another retrieval buffer as a fallback (UniProt Q9UBL3: nucleus; standard IHC practice). Compare intact nuclei with a no primary control, because prolonged heating can damage tissue and make an apparent gain in color difficult to interpret (standard IHC practice).
Could fixation explain weak or uneven ASH2L staining?
The selected ASH2L paraffin section caption does not state its fixative, so target specific fixation sensitivity is unknown (datasheet A06129-1). Record the fixative and fixation duration for each specimen, then compare sections processed together with the same EDTA retrieval at pH 8.0 (datasheet A06129-1; standard IHC practice). Examine nuclear detail and tissue preservation before interpreting a weak signal; damaged morphology makes cell level scoring unreliable (standard IHC practice). If fixation histories differ, use a consistently processed control tissue and report that difference as a limitation rather than attributing the staining change to ASH2L biology (standard IHC practice).
What staining pattern should count as ASH2L positive?
Score ASH2L primarily in nuclei: UniProt places the protein in the nucleus, and HPA tissue IHC describes mainly nuclear expression (UniProt Q9UBL3: nucleus; HPA tissue IHC). Use the counterstain to identify intact nuclei and assess chromogen within the relevant cell population, excluding folds and damaged areas (standard IHC practice). HPA ICC/IF reports nucleoplasm as the main location and an additional plasma membrane location, but a membrane rim in chromogenic tissue IHC needs independent validation before it is scored as positive (HPA subcellular; standard IHC practice). If color is predominantly cytoplasmic, check controls, retrieval, and detection background before changing the scoring compartment (UniProt Q9UBL3: nucleus; standard IHC practice).
Can a negative section rule out every ASH2L isoform?
No: UniProt lists 3 ASH2L isoforms, while the supplied caption gives no antibody epitope or isoform coverage (UniProt Q9UBL3: isoforms; datasheet A06129-1). Do not treat absence of chromogenic nuclear staining as evidence that every isoform is absent without an epitope map or independent assay (standard IHC practice). ASH2L has a B30.2/SPRY domain at residues 360–583 and reported modified residues, but those facts do not establish which epitope this antibody recognizes or how retrieval affects it (UniProt Q9UBL3: domains and modified residues). When results conflict, compare an independently validated antibody with a different known epitope on matched sections and document its specificity (standard IHC practice).
How should I check an ASH2L signal by multiplex IF?
For a separate IF/ICC experiment, pair ASH2L with a marker that identifies the cell population being assessed, and score nuclear overlap within those marked cells (UniProt Q9UBL3: nucleus; standard IF practice). Select fluorophores after checking tissue autofluorescence and channel bleed through, using a spectrally separated channel for the weaker signal (standard IF practice). Because the expected main signal is nucleoplasmic, include permeabilisation sufficient to give antibody access inside cells and compare it with a no primary control (HPA subcellular: nucleoplasm; standard IF practice). HPA also reports an additional plasma membrane location in ICC/IF; verify any membrane signal separately before transferring that interpretation to paraffin section IHC (HPA subcellular; standard IHC practice).
How can I reduce diffuse brown staining without losing nuclear signal?
First compare the ASH2L slide with a no primary control and inspect whether brown color follows intact nuclei or spreads through stroma and damaged areas (UniProt Q9UBL3: nucleus; standard IHC practice). The selected caption used 10% goat serum blocking, 2 μg/ml primary overnight at 4°C, and a peroxidase linked detection system with DAB (datasheet A06129-1). Include a peroxidase block, optimize blocking and washes, and shorten chromogen development if the control also turns brown (standard IHC practice). If background persists, compare a lower primary concentration on adjacent sections while retaining a known positive control; avoid calling weaker global color improved specificity without preserved nuclear signal (standard IHC practice).
How should I quantify ASH2L across differently cellular sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the scored cell population and nuclear compartment before comparing sections, because ASH2L is mainly nuclear and HPA reports different staining levels across cell populations (UniProt Q9UBL3: nucleus; HPA tissue IHC). Report the percentage of positive nuclei and intensity, or calculate a nuclear H-score from intensity categories 0–3 for a possible range of 0–300 (standard IHC practice). Normalize counts to the number of evaluable nuclei in the defined population, or report positive cell density per mm² of viable tissue (standard IHC practice). Apply one threshold and matched imaging conditions throughout, and exclude necrosis, folds, and section edges from the denominator (standard IHC practice).
When is an apparent ASH2L positive result likely to be artefactual?
A credible result follows intact nuclei in the specified cell population and exceeds staining in a no primary control (UniProt Q9UBL3: nucleus; standard IHC practice). Treat staining confined to section edges, folds, or necrotic areas as suspect, and check whether endogenous peroxidase or excess DAB development could explain brown color (standard IHC practice). HPA reports medium staining in kidney tubule cells but no detection in lung alveolar cells, so compare like cell populations rather than interpreting every tissue difference as an assay failure (HPA tissue IHC). HPA also rates its tissue staining Approved while noting low consistency with RNA expression; describe discordance without claiming that either measurement alone establishes specificity (HPA tissue IHC).
Boster reagents
Best ASH2L / Set1/Ash2 histone methyltransferase complex subunit ASH2 IHC Antibodies
A06129-1 and M06129 list human, mouse and rat reactivity. Both have human paraffin-section IHC images; A06129-1 also has a HELA cell IF/ICC image (catalog reactivity and 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 human lung adenocarcinoma and placenta paraffin sections, plus an IF/ICC image from HELA cells (catalog image captions). M06129 has an IHC image from a human kidney paraffin section and lists IF/ICC as an application (catalog image caption; catalog applications).
Which to pick: For tissue IHC, choose A06129-1 if its captioned EDTA pH 8.0 retrieval and 2 μg/ml primary condition are useful starting points; choose M06129 if a rabbit monoclonal with a human kidney paraffin-section image is preferred (A06129-1 IHC caption; M06129 catalog host/clone and IHC caption). For IF/ICC, A06129-1 has a HELA cell image at 5 μg/ml, while M06129 lists IF/ICC at 1:50 without an IF image (catalog IF images and dilutions). Both list human, mouse and rat reactivity, although the supplied IHC images show human tissue only; neither IHC caption reports the fixative (catalog reactivity and IHC 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 in 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 paraffin-section fixation consistent (standard IHC practice; not target-specific); Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A06129-1); Caveat: Staining has low concordance with RNA expression (HPA tissue IHC); Isoform / epitope: 3 isoforms; epitope differences unspecified (UniProt); Regulation: Higher expression in adult heart and testis (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.