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SIAH2 / E3 ubiquitin-protein ligase SIAH2 · IHC design guide
Design Immunohistochemistry for SIAH2
Plan chromogenic SIAH2 IHC in paraffin sections with the catalog antibody at 1:50–1:200 (datasheet: IHC dilution). Use testis spermatogonia as a positive reference and adipocytes as an unstained comparison, while accounting for low staining and RNA concordance (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
SIAH2 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 tissue staining observed; cytoplasm expected (HPA tissue IHC; UniProt) | |
| Staining pattern | Nuclear staining in several tissue types (HPA tissue IHC) | |
| Antigen retrieval | Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen) | |
| Positive control | Testis+4 more · see all | |
| Negative control | Adipose tissue+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 | Staining and RNA expression show low consistency (HPA tissue IHC) | |
| Regulation | Widely expressed at low level (UniProt) | |
| Isoform / epitope | No annotated isoforms; single 1–324 chain (UniProt) |
Section 1
Recommended SIAH2 IHC & IF Protocols
Compare the catalog antibody’s IHC-P protocol with published SIAH2 IHC protocols from prostate and lung cancer specimens (PMC8085430; PMC4651316; PMC4102802).
| Sample | Paraffin-embedded human colorectal carcinoma tissue; fixative not specified (datasheet A03379) |
| Fixation | Image fixative and duration unreported (datasheet A03379); 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-SIAH2, 1:50-1:200 (datasheet A03379) |
| 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 | SIAH2-positive staining in spermatogonia cells of testis (HPA tissue IHC: High). HPA tissue profile: Nuclear expression in several different tissue types. No signal in the no-primary control. |
💡Decision noteStart with Tris-EDTA pH 9.0 HIER at 95–98 °C for 20 min (page antigen-retrieval rule); compare EDTA pH 8.0 if staining is weak (PMC4651316).
Section 2
What Is the Expected SIAH2 Staining Pattern?
In paraffin-section IHC, expect chiefly nuclear staining in selected cells, including high staining in testicular spermatogonia and medium staining in glandular cells of the appendix and colon (HPA tissue IHC). SIAH2 is predominantly cytoplasmic and partly nuclear, with no transmembrane segment (UniProt O43255). Treat the tissue pattern cautiously: HPA rates its IHC reliability Approved but reports low consistency with RNA expression (HPA tissue IHC).
| Distinct nuclear staining in spermatogonia, with weaker or absent staining in neighboring cells. | This fits the high spermatogonial signal reported in testis (HPA tissue IHC). Score the identified cell population and nuclear compartment, rather than treating the whole section as uniformly positive (general IHC practice). The match supports interpretation but does not independently prove antibody specificity (general IHC practice). |
| Nuclear signal in appendix or colon glandular cells; some cytoplasmic signal may also be visible. | HPA reports medium staining in these glandular cells and describes nuclear expression across several tissues (HPA tissue IHC). Cytoplasmic signal is biologically plausible because UniProt describes SIAH2 as predominantly cytoplasmic and partly nuclear (UniProt O43255). Assess whether any cytoplasmic signal is cell-restricted and above background (general IHC practice). |
| Staining confined to cell borders, extracellular material, or an otherwise unexpected compartment. | This is discordant with the reported nuclear IHC pattern and cytoplasmic or nuclear localisation (HPA tissue IHC; UniProt O43255). SIAH2 has no transmembrane segment (UniProt O43255). Consider artefact or off-target staining, then check morphology and staining controls before assigning the signal to SIAH2 (general IHC practice). |
| Strong staining in adipocytes or cardiomyocytes, where HPA reports no detected signal. | The cell-type mismatch warrants scrutiny; HPA reports these cells as not detected, while also noting limited agreement between antibody staining and RNA data (HPA tissue IHC). Cross-reactivity or endogenous detection activity are possible explanations, not established diagnoses; compare no-primary and detection controls (general IHC practice). |
| Haze across the section obscures cell boundaries and nuclei. | Diffuse background cannot establish the cell-restricted pattern reported by HPA (HPA tissue IHC). It may arise from nonspecific antibody binding, detection activity, or incomplete washing (general IHC practice). Interpret staining only after background is low enough to distinguish positive cells from nearby tissue (general IHC practice). |
💡Expected SIAH2 appearanceA convincing IHC positive shows discernible nuclear staining in spermatogonia at high intensity, with cell-restricted signal above background; uniform haze or staining limited to extracellular spaces is suspect (HPA tissue IHC; general IHC practice).
| Tissue and cell selection | Testicular spermatogonia provide a reported high signal; appendix, colon and other listed positive cell populations show medium signal (HPA tissue IHC). Use the named cells when judging a section, since HPA levels do not apply uniformly to every cell in a tissue (HPA tissue IHC). |
| Compartment evidence | HPA describes nuclear tissue IHC, while UniProt describes predominantly cytoplasmic, partly nuclear localisation (HPA tissue IHC; UniProt O43255). Record nuclear and cytoplasmic scores separately when both appear (general IHC practice); do not force agreement between the two sources. |
| Antibody evidence | The HPA tissue antibody CAB069930 has an Approved IHC label, alongside a low-consistency warning for staining versus RNA (HPA tissue IHC; HPA antibodies). This supports cautious use of the reported pattern, not an Enhanced or independently reproduced IHC claim (HPA antibodies; general IHC practice). |
| Antigen retrieval and fixation | Target-specific fixation effects are not established by the supplied assay evidence. Verify with a matched IHC source before attributing a result to fixation. |
| IF/ICC Q&A: where should signal appear? | HPA reports mainly nucleoplasmic signal, with additional vesicles, in ICC-IF; HPA061293 is Supported for ICC (HPA subcellular ICC-IF; HPA antibodies). That observation informs an IF/ICC interpretation, but it does not establish a paraffin-section IHC pattern or supply an IF/ICC protocol (HPA tissue IHC; HPA subcellular ICC-IF). |
| Situation | Likely cause | Next action |
|---|---|---|
| No staining in testicular spermatogonia. | The expected high cell population may be absent from the field, or the IHC workflow may have failed (HPA tissue IHC; general IHC practice). | First locate spermatogonia on the counterstained section; then check the positive-control section, antibody application, retrieval, and detection steps against the IHC-P instructions (general IHC practice). |
| Nuclei and cytoplasm both stain. | HPA's tissue IHC description favors nuclei, whereas UniProt allows cytoplasmic and nuclear SIAH2 (HPA tissue IHC; UniProt O43255). Background can also blur compartment boundaries (general IHC practice). | Score each compartment separately and compare signal with adjacent cells and the no-primary control (general IHC practice). Report the source discrepancy rather than calling all cytoplasmic staining artefactual. |
| Adipocytes or cardiomyocytes stain strongly. | These cells are listed as not detected by HPA; off-target binding or endogenous detection activity may explain unexpected color (HPA tissue IHC; general IHC practice). | Verify cell identity and compare no-primary and detection controls (general IHC practice). If those controls are clean, treat the result as an unresolved disagreement with HPA and seek independent specificity evidence. |
| Diffuse chromogen obscures the expected cells. | Nonspecific binding, incomplete washing, or endogenous detection activity may raise section-wide background (general IHC practice). | Inspect no-primary and detection controls, review blocking and wash steps, and optimize the IHC-validated antibody under its IHC-P instructions (general IHC practice). Re-score only when nuclei and cell boundaries are discernible. |
| Signal appears only on cell borders or outside cells. | This distribution conflicts with HPA's nuclear tissue pattern and UniProt's cytoplasmic or nuclear localisation; no transmembrane segment is annotated (HPA tissue IHC; UniProt O43255). | Check section morphology, counterstain, no-primary control, and detection background (general IHC practice). Do not count border-only or extracellular color as a SIAH2-positive cell without independent support. |
| An HPA negative cell population stains faintly. | HPA's not detected calls describe its observations, and its tissue IHC record warns of low consistency with RNA data (HPA tissue IHC). Faint color may also be background (general IHC practice). | Compare intensity and compartment with a reported positive population and a no-primary control (HPA tissue IHC; general IHC practice). Record the discrepancy without converting a faint signal into a confirmed positive result. |
Experiment evidence
IHC & IF images showing SIAH2 staining
A03379 · IHCImmunohistochemistry (IHC) analyzes of Siah-2 (E266) pAb in paraffin-embedded human colorectal carcinoma tissue at 1:50.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000181788-SIAH2/tissue/cerebral+cortex#imid_19000309
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000181788-SIAH2/tissue/colon#imid_19000227
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000181788-SIAH2/tissue/liver#imid_19000228
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000181788-SIAH2/tissue/kidney#imid_19000245
HPA · ICC-IF Hep-G2Human Protein Atlas immunofluorescence, Hep-G2, antibody HPA061293; green = SIAH2, blue = nucleus, red = microtubules.
HPA · ICC-IF Hep-G2Human Protein Atlas immunofluorescence, Hep-G2, antibody HPA061293; green = SIAH2, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for SIAH2 IHC & IF
🧪Run testis first; spermatogonia should stain (HPA: High in spermatogonia). Use adipose tissue adipocytes as the negative tissue (HPA: Not detected in adipocytes); on the testis slide, cells without staining should remain at background, though HPA does not identify a validated internal-negative cell type there (HPA: testis row).
Positive control tissue: Testis (Spermatogonia cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show SIAH2 in Hep-G2, MCF-7, U2OS, RPTEC/TERT1, hTERT-RPE1 (serum starved), with annotated localisation: Nucleoplasm (supported) (HPA subcellular).
Technical controls: Include no-primary (secondary-only) and host-species- and immunoglobulin-matched isotype controls, plus a matched SIAH2-knockout sample or peptide-block control if available (standard IHC practice). For chromogenic testis sections, quench endogenous peroxidase and inspect the control slide for residual background (standard IHC practice).
⚠️Feasibility: A target-specific fixation window and its effect are unreported, and the selected A03379 paraffin-section caption does not state a fixative (selected-SKU IHC caption). Retrieval dependency is unreported; optimize antigen retrieval on the testis positive control before scoring experimental sections (standard paraffin IHC practice; HPA: High in spermatogonia). The supplied evidence does not establish that frozen sections or IF are easier, or identify a testis-specific artefact (HPA: tissue and subcellular entries; selected-SKU IHC caption).
HPA tissue IHC evidence for SIAH2
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 |
|---|---|---|---|---|
| Testis | Spermatogonia cells | High | Protein (IHC) | HPA → |
| Appendix | Glandular cells | Medium | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | Medium | Protein (IHC) | HPA → |
| Bronchus | Basal cells | Medium | Protein (IHC) | HPA → |
| Cervix | Squamous epithelial cells | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Caudate | Glial cells | Not detected | Protein (IHC) | HPA → |
| Cerebellum | Cells in granular layer | Not detected | Protein (IHC) | HPA → |
| Cerebral cortex | Endothelial cells | Not detected | Protein (IHC) | HPA → |
| Heart muscle | Cardiomyocytes | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced SIAH2 IHC Tips
Troubleshoot SIAH2 staining in paraffin sections by checking retrieval, compartment, cell type, and controls before comparing chromogenic signal across samples.
How should I retrieve SIAH2 in paraffin sections with weak nuclear staining?
Start with Tris-EDTA pH 9.0 HIER at 95–98 °C for 20 min (page retrieval setting: nuclear antigen). Cool sections in retrieval buffer and process control sections alongside test sections so heating differences do not confound staining (standard IHC practice). If staining remains weak, compare citrate pH 6.0 as a fallback on serial sections while holding antibody dilution and detection constant (standard IHC practice). Assess nuclear and cytoplasmic staining because SIAH2 occupies both compartments (UniProt O43255 subcellular location). The catalog image documents 1:50 staining in a paraffin section but does not establish which retrieval method was used (caption A03379).
Can I attribute weak SIAH2 staining to fixation?
Target-specific sensitivity of SIAH2 to fixation is unknown from the supplied evidence (caption A03379; HPA tissue IHC). The catalog image shows paraffin-embedded human colorectal carcinoma at 1:50, but its fixative and fixation duration are unreported (caption A03379). Record those variables for new blocks and compare matched sections processed with the same retrieval and detection conditions (standard IHC practice). Include a positive control section in each staining run to separate processing failure from a low signal in the sample (standard IHC practice). Do not infer fixation sensitivity from SIAH2 phosphorylation, topology, or the HPA tissue staining pattern (UniProt O43255; HPA tissue IHC).
Should I score SIAH2 in nuclei, cytoplasm, or both?
Score nuclear and cytoplasmic staining separately because SIAH2 is reported in both compartments, with predominantly cytoplasmic localisation in UniProt (UniProt O43255 subcellular location). Tissue IHC instead reports nuclear expression across several tissue types (HPA tissue IHC). HPA cell imaging supports nucleoplasmic localisation and also reports vesicles (HPA subcellular). On chromogenic sections, use a counterstain and consistent compartment boundaries before assigning a cell as positive (standard IHC practice). Report compartment, intensity, and cell type together; a single pooled score could hide differences between nuclear and cytoplasmic staining (UniProt O43255 subcellular location; HPA tissue IHC).
How can I investigate unexpected SIAH2 staining when the antibody epitope is unspecified?
The supplied record annotates 0 SIAH2 isoforms, while the antibody epitope is unspecified (UniProt O43255 isoforms; caption A03379). SIAH2 has a single annotated chain spanning residues 1–324 and several phosphorylated residues, including positions 6, 16, 26, 28, and 68 (UniProt O43255 processing; modified residues). Without epitope mapping, a change in staining cannot be assigned to any one modification (UniProt O43255 modified residues; caption A03379). Compare staining after SIAH2 depletion or with an independently validated antibody, using matched sections and scoring rules (standard IHC validation practice). Functional overlap with SIAH1 does not establish antibody cross-reactivity (UniProt O43255 function).
How should I check an IF result against this chromogenic SIAH2 guide?
Treat IF or ICC as a separately optimised assay; the supplied catalog example documents paraffin-section IHC only (caption A03379). For a tissue comparison, multiplex SIAH2 with a validated spermatogonia marker because HPA reports high staining in those cells (HPA tissue IHC: testis, spermatogonia cells). Choose a far-red fluorophore when shorter-wavelength tissue autofluorescence obscures signal, and include single-channel controls (standard IF practice). Permeabilise for an intracellular epitope because SIAH2 has no transmembrane segment and is reported in cytoplasm and nucleus (UniProt O43255 topology; subcellular location). Optimise permeabilisation on control material; the antibody epitope and IF fixation conditions are not supplied (caption A03379).
What should I adjust if SIAH2 DAB staining is diffuse?
Run a no-primary control to check secondary-reagent and chromogen background in the same tissue (standard chromogenic IHC practice). Apply an endogenous peroxidase block before horseradish peroxidase detection, then compare matched sections developed for the same time (standard chromogenic IHC practice). Titrate the primary antibody and use a protein block if diffuse staining persists; 1:50 is the catalog image condition, not a supplied optimum for every tissue (caption A03379; standard IHC practice). Inspect tissue edges and damaged areas separately because they can collect nonspecific stain (standard IHC practice). Evaluate nuclear and cytoplasmic patterns against controls, since both compartments are reported for SIAH2 (UniProt O43255 subcellular location).
How should I quantify SIAH2 across stained tissue sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and score nuclear and cytoplasmic DAB signal separately before comparing sections (standard IHC scoring practice; UniProt O43255 subcellular location). An H-score combines percentages at intensity levels 0–3 and spans 0–300; record percent-positive cells as well (standard IHC scoring practice). For spatial comparisons, report positive cells per mm² of analysable tissue and exclude necrotic or damaged areas using the same rules throughout (standard IHC scoring practice). Normalise cell counts to the relevant cell population or analysable area, and keep counterstain and threshold settings consistent (standard IHC scoring practice). State the compartment and denominator with every result (standard IHC scoring practice).
How can I distinguish genuine SIAH2 staining from artefact?
Look for reproducible cellular staining in the expected nuclear or cytoplasmic compartments, while recording which compartment predominates (UniProt O43255 subcellular location; HPA tissue IHC). High signal in spermatogonia is supported by HPA, whereas adipocytes and cardiomyocytes were reported as not detected in its tissue panel (HPA tissue IHC). Treat those patterns as comparison points rather than absolute controls because HPA reports low consistency between antibody staining and RNA expression (HPA tissue IHC reliability). Check whether signal follows section edges, necrotic regions, or the no-primary control (standard chromogenic IHC practice). Residual endogenous peroxidase can produce DAB signal, so review the peroxidase-block control before calling weak staining positive (standard chromogenic IHC practice).
Boster reagents
Best SIAH2 / E3 ubiquitin-protein ligase SIAH2 IHC Antibodies
The IHC-validated SIAH2 antibody has real staining data from human paraffin-embedded colorectal carcinoma tissue (IHC image caption) and listed Human, Mouse, and Rat reactivity (catalog: A03379).
Real IHC data
Anti-Siah-2 (E266) Antibody
Cat # A03379
A03379 is listed for IHC with Human, Mouse, and Rat reactivity (catalog: A03379). Its IHC figure shows human paraffin-embedded colorectal carcinoma tissue at 1:50 (IHC image caption: A03379).
Which to pick: Choose A03379 for paraffin-section tissue IHC: its figure shows human colorectal carcinoma tissue, and the catalog lists an IHC dilution of 1:50–1:200 (IHC image caption and catalog: A03379). The caption does not report a fixative (IHC image caption: A03379). For cross-species work, A03379 lists Mouse and Rat reactivity alongside Human, with a Rabbit host and no clone specified (catalog: A03379); no IF/ICC option is listed (catalog: A03379 applications).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry O43255 (SIAH2_HUMAN, E3 ubiquitin-protein ligase SIAH2).
- Human Protein Atlas. SIAH2 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. SIAH2 subcellular location (ICC-IF): Mainly localized to the nucleoplasm. In addition localized to vesicles..
- Human Protein Atlas. SIAH2 antibody validation summary (2 antibodies).
- Targeting ADT-Induced Activation of the E3 Ubiquitin Ligase Siah2 to Delay the Occurrence of Castration-Resistant Prostate Cancer. Frontiers in oncology 2021 — PMC8085430.
- The Expression of the Ubiquitin Ligase SIAH2 (Seven In Absentia Homolog 2) Is Increased in Human Lung Cancer. PloS one 2015 — PMC4651316.
- SIAH2 antagonizes TYK2-STAT3 signaling in lung carcinoma cells. Oncotarget 2014 — PMC4102802.
- Expression of the preadipocyte marker ZFP423 is dysregulated between well-differentiated and dedifferentiated liposarcoma. BMC cancer 2022 — PMC8939188.
- PubMed PMID:9403064 — UniProt-cited evidence.
- PubMed PMID:10207103 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for SIAH2 (E3 ubiquitin-protein ligase SIAH2). Expected localisation Nuclear tissue staining observed; cytoplasm expected (HPA tissue IHC; UniProt). Antigen retrieval: Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen). Validated IHC antibody: A03379 at 1:50-1:200 (the product's own IHC validation image is shown). Controls (HPA): positive Testis (Spermatogonia cells); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human colorectal carcinoma tissue; fixative not specified (datasheet A03379); 2. Fixation: Image fixative and duration unreported (datasheet A03379); 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-SIAH2, 1:50-1:200 (datasheet A03379); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across sections. (standard IHC practice; not target-specific); Caveat: Staining and RNA expression show low consistency (HPA tissue IHC); Isoform / epitope: No annotated isoforms; single 1–324 chain (UniProt); Regulation: Widely expressed at low level (UniProt). Evidence: UniProt O43255, HPA tissue IHC · Approved, HPA ICC-IF · Mainly localized to the nucleoplasm. In addition localized to…, Datasheet A03379, PMC8085430, PMC4651316. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/SIAH2/immunohistochemistry-ihc-guide.