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PIAS4 / E3 SUMO-protein ligase PIAS4 · IHC design guide
Design Immunohistochemistry for PIAS4
Plan PIAS4 chromogenic IHC on paraffin sections using round or early spermatids as a high-staining reference (HPA tissue IHC). The catalog antibody starts at 5 μg/mL (datasheet); interpret cytoplasmic tissue staining alongside the nuclear/PML-body annotation (HPA tissue IHC; UniProt).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
PIAS4 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 | Cytoplasmic in tissue (HPA tissue IHC); nuclear/PML body (UniProt) | |
| Staining pattern | Cytoplasmic staining; high in round/early spermatids (HPA tissue IHC) | |
| Antigen retrieval | Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen) | |
| Positive control | Testis | |
| 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); Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A02994) | |
| Caveat | Tissue staining is cytoplasmic despite nuclear annotation (HPA tissue IHC; UniProt) | |
| Regulation | Highest expression in testis (UniProt) | |
| Isoform / epitope | No listed isoforms; mature chain spans residues 2–510 (UniProt) |
Section 1
Recommended PIAS4 IHC & IF Protocols
The catalog antibody's IHC-P protocol is accompanied by two published PIAS4 IHC workflows (PMC12853826; PMC11954794).
| Sample | Tissue sections; selected-image fixative not specified (standard IHC workflow) |
| Fixation | Image fixative and duration unreported (datasheet A02994); 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-PIAS4, 5 μg/mL (datasheet A02994) |
| 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 | PIAS4-positive staining in round or early spermatids of testis (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression in several tissues, including testis. No signal in the no-primary control. |
💡Decision noteStart with Tris-EDTA pH 9.0 retrieval at 95–98 °C for 20 min (page antigen-retrieval rule; UniProt: nuclear PIAS4).
Section 2
What Is the Expected PIAS4 Staining Pattern?
PIAS4 is a nuclear protein with no transmembrane segment (UniProt Q8N2W9). For paraffin-section IHC, expect the strongest documented staining in testicular round or early spermatids, although HPA describes cytoplasmic staining across several tissues (HPA: testis High; tissue IHC profile). HPA rates the tissue IHC evidence Enhanced while describing only medium consistency with RNA expression (HPA: reliability description).
| Strong cytoplasmic staining in round or early spermatids on a testis section. | This matches the reported high testis signal and cytoplasmic tissue IHC profile (HPA: testis High; tissue IHC profile). Record the stained cell type and compartment separately; a nuclear location is also reported for PIAS4 (UniProt Q8N2W9). |
| Nuclear staining, possibly appearing as discrete foci, accompanies staining in the expected cells. | A nuclear signal is biologically plausible: PIAS4 localizes to the nucleus and a subset of PML bodies (UniProt Q8N2W9). HPA’s nucleoplasm result comes from ICC-IF, so it does not establish the appearance or required intensity of nuclear foci in chromogenic IHC (HPA: subcellular ICC-IF). |
| The apparent positive signal is confined to cell membranes, with no expected spermatid staining. | An exclusively membranous pattern conflicts with the reported nucleus and PML-body location and lack of a transmembrane segment (UniProt Q8N2W9). Treat it as suspect; review morphology, controls and detection background before assigning PIAS4 positivity (general IHC practice). |
| Strong staining dominates HPA-listed negative cell populations, such as adipocytes or bone-marrow hematopoietic cells. | These populations were reported as not detected (HPA: adipocytes and bone-marrow hematopoietic cells). Unexpected signal may reflect cross-reactivity or endogenous detection activity; it is not, by itself, proof of either cause (general IHC practice). |
| Weak, widespread color obscures cell boundaries, or testis round or early spermatids show no signal. | Diffuse background makes compartment scoring unreliable (general IHC practice). Absent signal in the reported high-staining testis population calls for a technical review, but HPA’s Enhanced rating and medium RNA–staining consistency do not guarantee positivity in every specimen (HPA: testis High; reliability description). |
💡Expected PIAS4 appearanceCall a result supportive when round or early spermatids show clear, strong staining consistent with HPA’s cytoplasmic testis profile (HPA: testis High; tissue IHC profile); isolated membrane staining or color distributed across negative cell types is suspect (UniProt Q8N2W9 topology; HPA: negative tissue IHC populations).
| Cell type and tissue | Testis round or early spermatids are the supplied high-staining benchmark (HPA: testis High). HPA also reports several low or undetected populations; do not score an entire tissue as uniformly positive or negative (HPA: tissue IHC). |
| Compartment evidence | HPA describes cytoplasmic tissue IHC, whereas UniProt places PIAS4 in the nucleus and PML bodies (HPA: tissue IHC profile; UniProt Q8N2W9). Keep assay-specific observations distinct when judging a slide. |
| Antibody validation | HPA010598 is IHC Enhanced; HPA076008 has no listed IHC rating (HPA: antibody validation). An Enhanced rating supports the reported pattern but does not authenticate every stained structure (HPA: reliability description). |
| Topology and processing | PIAS4 has no transmembrane segment or signal peptide, and its annotated chain is residues 2–510 (UniProt Q8N2W9). These annotations provide no basis for expecting a membrane or secreted staining pattern. |
| Retrieval and fixation evidence | No target-specific retrieval condition or fixation sensitivity is supplied (UniProt Q8N2W9; HPA: tissue IHC). Evaluate retrieval as a general paraffin IHC variable without claiming a PIAS4-specific effect (general IHC practice). |
| IF/ICC comparison | HPA supports nucleoplasm and approves an additional centrosome location, while both listed antibodies have Uncertain ICC ratings (HPA: subcellular ICC-IF; antibody validation). Use that evidence to interpret IF, not to prescribe an IF protocol. |
| Situation | Likely cause | Next action |
|---|---|---|
| No signal in testis round or early spermatids. | The expected high-staining population is absent from the result (HPA: testis High); the available evidence does not identify a PIAS4-specific fixation or retrieval cause. | Check that the relevant cells are present, then review primary-antibody application, detection reagents and a known-positive control; assess retrieval using a standard paraffin IHC workflow (general IHC practice). |
| Weak brown color covers most cells or extracellular space. | Diffuse deposition can obscure the reported cell-specific pattern (HPA: testis High; general IHC practice); appearance alone does not identify its source. | Compare a section processed without primary antibody, then inspect blocking, washes and chromogen development before adjusting working conditions (general IHC practice). |
| Strong color appears in adipocytes or bone-marrow hematopoietic cells. | HPA reports these cell populations as not detected (HPA: tissue IHC). Cross-reactivity and endogenous detection activity are possibilities, not established explanations (general IHC practice). | Check the no-primary control and the tissue distribution on the same run; confirm any unexpected positive call with an independently validated antibody where available (general IHC practice). |
| Staining appears only on cell membranes. | Exclusive membrane staining is inconsistent with the annotated nuclear location and absence of a transmembrane segment (UniProt Q8N2W9). | Recheck cell boundaries against the counterstain and inspect background controls; withhold a PIAS4-positive interpretation until the pattern is independently supported (general IHC practice). |
| Nuclear staining differs from the cytoplasmic tissue IHC profile. | The sources describe different assay readouts: cytoplasmic tissue IHC and nuclear or PML-body localization (HPA: tissue IHC profile; UniProt Q8N2W9). | Report the observed compartment and cell type. Compare with the reported testis pattern and antibody validation; do not require visible PML foci in chromogenic sections (HPA: testis High; HPA010598 IHC Enhanced; general IHC practice). |
| What should an IF/ICC result show? | Nucleoplasm is the supported main location and centrosome an approved additional location, but the listed ICC antibody ratings are Uncertain (HPA: subcellular ICC-IF; antibody validation). | Interpret nuclear signal as compatible with PIAS4 localization, assess any centrosome signal cautiously, and use appropriate IF controls before claiming specificity (HPA: subcellular ICC-IF; general IF practice). |
Experiment evidence
IHC & IF images showing PIAS4 staining
A02994 · IHCImmunohistochemistry of PIAS4 in rat brain tissue with PIAS4 antibody at 5 μg/mL.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000105229-PIAS4/tissue/cerebral+cortex#imid_2863716
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000105229-PIAS4/tissue/colon#imid_2863658
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000105229-PIAS4/tissue/liver#imid_2863659
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000105229-PIAS4/tissue/kidney#imid_2863644
HPA · ICC-IF U-251MGHuman Protein Atlas immunofluorescence, U-251MG, antibody HPA010598; green = PIAS4, blue = nucleus, red = microtubules.
HPA · ICC-IF U-251MGHuman Protein Atlas immunofluorescence, U-251MG, antibody HPA010598; green = PIAS4, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for PIAS4 IHC & IF
🧪Run testis first: round or early spermatids should show PIAS4 staining (HPA: High). Use adipose tissue as the negative tissue, with adipocytes expected to lack detectable staining (HPA: Not detected); on the testis slide, treat non-spermatid cells as internal negative comparators only if they show no nuclear signal, since their PIAS4 status is not specified by the supplied HPA row.
Positive control tissue: Testis (Round or early spermatids, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show PIAS4 in U-251MG, U2OS, HEK293, SH-SY5Y, with annotated localisation: Nucleoplasm (supported) (HPA subcellular).
Technical controls: Include a no-primary (secondary-only) control, a host-species- and clonality-matched isotype control, and PIAS4-knockout material as a biological negative (standard IHC practice). Quench endogenous peroxidase and inspect the testis section for background chromogen before scoring nuclear staining (standard chromogenic IHC practice).
⚠️Feasibility: No target-specific fixation window or retrieval dependency is reported in the supplied evidence; optimize antigen retrieval empirically for paraffin sections. Frozen sections or IF/ICC may avoid paraffin retrieval, but the supplied evidence does not establish that either is easier; dense testis cell populations require careful assignment of staining to round or early spermatids (HPA: High in round or early spermatids). The selected A02994 rat-brain tissue-IHC caption reports 5 µg/mL but leaves the fixative unreported (selected tissue-IHC caption: fixative not stated).
HPA tissue IHC evidence for PIAS4
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — Medium consistency between antibody staining and RNA expression data.). Rows are taken directly from the HPA tissue chart — click any row's HPA link to view the source.
Positive expression · recommended positive controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Testis | Round or early spermatids | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Caudate | Glial cells | Not detected | Protein (IHC) | HPA → |
| Cerebellum | Cells in granular layer | Not detected | Protein (IHC) | HPA → |
| Duodenum | Glandular cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced PIAS4 IHC Tips
Troubleshoot PIAS4 staining in paraffin sections by checking retrieval, cell identity and subcellular pattern before interpreting chromogenic signal.
Which retrieval conditions should I try first for PIAS4 in paraffin sections?
Start with Tris-EDTA pH 9.0 heat-induced retrieval at 95–98 °C for 20 minutes (page retrieval specification). Allow sections to cool in retrieval buffer before proceeding, and compare runs using the same heating and cooling schedule (standard IHC practice). PIAS4 is chiefly nuclear, so assess whether staining resolves within nuclei rather than judging retrieval by total brown signal alone (UniProt: nucleus and PML body; standard IHC practice). If staining remains weak, test citrate pH 6.0 as a fallback on matched sections, recording any change in morphology or background (standard IHC practice). Keep the antibody concentration and detection steps constant during that comparison (standard IHC practice).
Could fixation explain weak or uneven PIAS4 staining?
PIAS4-specific sensitivity to fixation is unknown from the supplied evidence; the rat brain IHC caption reports 5 µg/mL antibody but does not state a fixative (A02994 tissue-IHC caption). Record the actual fixative, fixation duration and section handling for each specimen before comparing signal across blocks (standard IHC practice). On matched material, compare a consistent fixation schedule and the specified Tris-EDTA pH 9.0 retrieval while holding detection conditions constant (page retrieval specification; standard IHC practice). If nuclear detail and staining both vary across a section, inspect processing and section adhesion before attributing the pattern to PIAS4 biology (standard IHC practice). HPA tissue patterns do not establish a fixation effect (HPA: tissue IHC profile).
Should PIAS4 stain nuclei or cytoplasm in tissue IHC?
Expect a nuclear component because PIAS4 is assigned to the nucleus and PML bodies, while HPA cell imaging supports nucleoplasmic localization (UniProt: subcellular location; HPA: subcellular). HPA tissue IHC also reports cytoplasmic expression across several tissues, including testis, with only medium agreement between staining and RNA expression (HPA: tissue IHC profile; HPA: Enhanced reliability description). Score nuclear and cytoplasmic compartments separately rather than treating every brown area as equivalent PIAS4 signal (standard IHC practice). Compare staining with hematoxylin-defined nuclei and tissue morphology, then inspect whether cytoplasmic signal persists outside damaged or folded regions (standard IHC practice). A disagreement between compartments warrants antibody-specific validation rather than an assumed biological relocalization (standard IHC practice).
How do epitope uncertainty and PIAS4 annotations affect IHC interpretation?
The supplied record lists 0 isoforms, a SAP domain at residues 12–46, and a PINIT region at 119–279 (UniProt: isoforms and domains). It also lists acetylation at residues 2 and 114, but supplies no antibody epitope or evidence that either modification changes staining (UniProt: modified residues; supplied antibody evidence). Request the immunogen or epitope information before assigning a weak result to epitope masking (standard IHC practice). Compare retrieval conditions on matched sections while keeping antibody concentration and chromogenic detection fixed (standard IHC practice). The lack of annotated isoforms does not establish that every observed staining difference represents a PIAS4 modification (UniProt: isoforms; standard IHC interpretation).
How should I check PIAS4 localization with multiplex IF?
For a separate IF experiment, pair PIAS4 with a validated marker for the cell population under study; round or early spermatids are a reported high-staining population in testis IHC (HPA: High in round or early spermatids). Add a nuclear counterstain to assess the expected nucleoplasmic pattern and possible nuclear foci (HPA: subcellular; UniProt: nucleus and PML body; standard IF practice). Choose a fluorophore channel away from the tissue's strongest autofluorescence, checking single-label and unstained controls before interpreting overlap (standard IF practice). PIAS4 has no transmembrane segment and is assigned mainly to the nucleus, so permeabilize fixed cells or sections sufficiently for intracellular antibody access (UniProt: topology and subcellular location; standard IF practice). Optimize IF conditions independently of the rat brain chromogenic IHC caption (A02994 tissue-IHC caption; standard IF practice).
What should I check when PIAS4 IHC has diffuse brown background?
First compare the stained section with a no-primary control and inspect whether diffuse color follows tissue edges, folds or damaged areas (standard IHC practice). Check endogenous peroxidase blocking before DAB development, because native enzyme activity can generate brown signal independently of antibody binding (standard chromogenic IHC practice). Titrate the IHC-validated antibody and review blocking, wash times and detection exposure together, changing one condition at a time (standard IHC practice). The selected rat brain image used 5 µg/mL, which is evidence for that image rather than a universal working concentration (A02994 tissue-IHC caption). Judge residual signal against nuclear morphology and the reported PIAS4 localization, while recognizing HPA's reported cytoplasmic tissue staining (UniProt: subcellular location; HPA: tissue IHC profile).
How should I quantify PIAS4 staining across tissue sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring; PIAS4 has nuclear localization evidence, whereas HPA tissue IHC also reports cytoplasmic staining (UniProt: subcellular location; HPA: tissue IHC profile). For nuclear staining, record the percentage of positive nuclei and an H-score using intensity categories 0–3 within a prespecified region (standard IHC scoring practice). If counting discrete positive cells, report density per mm² of viable tissue and normalize to the number of eligible cells or measured viable area (standard IHC quantification practice). Keep section thickness, retrieval, imaging and threshold rules consistent across comparison groups (standard IHC practice). Report nuclear and cytoplasmic results separately, with the number of specimens and fields analyzed (standard IHC reporting practice).
How can I distinguish convincing PIAS4 staining from artefact?
A convincing result should be reproducible in intact cells, with a nuclear component consistent with UniProt and HPA cell-imaging localization (UniProt: nucleus and PML body; HPA: nucleoplasm). In testis, high signal in round or early spermatids offers a reported cell-level reference, while several listed cell populations are not detected by HPA tissue IHC (HPA: tissue IHC positive and negative profiles). Treat staining restricted to section edges, folds or necrotic regions as suspect until it survives comparison with intact areas (standard IHC practice). Use a no-primary control and an appropriate peroxidase block to assess brown signal generated by the detection workflow (standard chromogenic IHC practice). Interpret persistent cytoplasmic staining cautiously because HPA reports it despite nuclear localization evidence (HPA: tissue IHC profile; UniProt: subcellular location).
Boster reagents
Best PIAS4 / E3 SUMO-protein ligase PIAS4 IHC Antibodies
A02994 has pictured PIAS4 IHC staining in rat brain; Human, Mouse and Rat reactivity is listed, with no IF image supplied (A02994 image caption; catalog reactivity; IF image alts).
Real IHC data
Anti-E3 SUMO-protein ligase PIAS4 PIAS4 Antibody
Cat # A02994
A02994 is listed for IHC-P and shows PIAS4 staining in rat brain at 5 μg/mL (catalog applications; A02994 image caption). Human and Mouse reactivity is listed, but neither has a supplied IHC image (catalog reactivity; A02994 image alts).
Which to pick: For tissue IHC, choose A02994: its own caption shows rat brain staining at 5 μg/mL, while its application list specifies IHC-P; the caption does not report the fixative or section processing (A02994 image caption; catalog applications). For IF/ICC, A02994 has a suggested IF starting concentration of 20 μg/mL, but no IF image or IF/ICC validation is supplied (catalog dilution; IF image alts; catalog applications). For cross-species work, A02994 lists Human, Mouse and Rat reactivity; its host is Rabbit and clonality is unreported (catalog reactivity; host; clone).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q8N2W9 (PIAS4_HUMAN, E3 SUMO-protein ligase PIAS4).
- Human Protein Atlas. PIAS4 tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. PIAS4 subcellular location (ICC-IF): Mainly localized to the nucleoplasm. In addition localized to the centrosome..
- Human Protein Atlas. PIAS4 antibody validation summary (2 antibodies).
- Down-regulation of EHMT2 through irisin-mediated epigenetic modification promotes osteogenesis via promoting DLX3 transcription. Cell communication and signaling : CCS 2026 — PMC12853826.
- Tanshinone IIA destabilizes SLC7A11 by regulating PIAS4-mediated SUMOylation of SLC7A11 through KDM1A, and promotes ferroptosis in breast cancer. Journal of advanced research 2025 — PMC11954794.
- Hypoxia Stimulates SUMOylation-Dependent Stabilization of KDM5B. Frontiers in cell and developmental biology 2021 — PMC8719622.
- COMMD7 Regulates NF-κB Signaling Pathway in Hepatocellular Carcinoma Stem-like Cells. Molecular therapy oncolytics 2019 — PMC6350112.
- PubMed PMID:9724754 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
- PubMed PMID:11388671 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for PIAS4 (E3 SUMO-protein ligase PIAS4). Expected localisation Cytoplasmic in tissue (HPA tissue IHC); nuclear/PML body (UniProt). Antigen retrieval: Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen). Validated IHC antibody: A02994 at 5 μg/mL (the product's own IHC validation image is shown). Controls (HPA): positive Testis (Round or early spermatids); negative Adipose tissue. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Tissue sections; selected-image fixative not specified (standard IHC workflow); 2. Fixation: Image fixative and duration unreported (datasheet A02994); 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-PIAS4, 5 μg/mL (datasheet A02994); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across sections (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 A02994); Caveat: Tissue staining is cytoplasmic despite nuclear annotation (HPA tissue IHC; UniProt); Isoform / epitope: No listed isoforms; mature chain spans residues 2–510 (UniProt); Regulation: Highest expression in testis (UniProt). Evidence: UniProt Q8N2W9, HPA tissue IHC · Enhanced, HPA ICC-IF · Mainly localized to the nucleoplasm. In addition localized to the…, Datasheet A02994, PMC12853826, PMC11954794. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/PIAS4/immunohistochemistry-ihc-guide.