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ACTN4 / Alpha-actinin-4 · IHC design guide
Design Immunohistochemistry for ACTN4
Plan ACTN4 chromogenic IHC in paraffin sections using the observed cytoplasmic tissue pattern (HPA tissue IHC). Compare staining across sections with consistent fixation, and account for the reported antibody specificity caution (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
ACTN4 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 | Predominantly cytoplasmic in tissue (HPA tissue IHC) | |
| Staining pattern | Cytoplasmic staining across essentially all tissue types (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet PB9974) | |
| Positive control | Colon+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 | Antibody staining may reflect more than one gene (HPA tissue IHC) | |
| Regulation | Blood-vessel-enhanced RNA expression (HPA tissue RNA) | |
| Isoform / epitope | 3 isoforms; epitope coverage is unspecified (UniProt) |
Section 1
Recommended ACTN4 IHC & IF Protocols
The catalog antibody’s IHC-P protocol is accompanied by published ACTN4 staining conditions for hepatocellular carcinoma (PMC8798106) and upper urinary tract urothelial carcinoma (PMC10394141).
| Sample | Paraffin-embedded human colonic adenocarcinoma tissue; fixative not specified (datasheet PB9974) |
| Fixation | Image fixative and duration unreported (datasheet PB9974); 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 PB9974); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet PB9974) |
| Primary antibody | Rabbit anti-ACTN4, 2-5μg/ml (datasheet PB9974) |
| Primary incubation | Overnight at 4 °C (datasheet PB9974) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet PB9974) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | ACTN4-positive staining in endothelial cells of colon (HPA tissue IHC: High). HPA tissue profile: Ubiquitous cytoplasmic expression in essentially all tissue types. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA pH 8.0 for the catalog antibody (datasheet PB9974). Use the cited citrate conditions when reproducing either published assay (PMC8798106; PMC10394141).
Section 2
What Is the Expected ACTN4 Staining Pattern?
ACTN4 is widely expressed and has no transmembrane segment (UniProt O43707). In paraffin-section IHC, expect predominantly cytoplasmic staining across many tissue types, with strong examples in colon endothelial cells, liver cholangiocytes, lung type II alveolar cells, placental trophoblastic cells, and bladder urothelial cells (HPA tissue IHC). HPA rates its tissue pattern Supported, with medium consistency between staining and RNA data, and cautions that the antibodies target proteins from more than one gene (HPA tissue IHC).
| Cytoplasmic chromogen is clear in colon endothelial cells or liver cholangiocytes, with cell boundaries and counterstained nuclei still readable (HPA tissue IHC). | This fits the reported High staining in those cells and ACTN4's cytoplasmic location (HPA tissue IHC; UniProt O43707). Score intensity in the identified cells; do not treat every stained cell in a widely expressing tissue as an equally strong reference (HPA tissue IHC). |
| Signal appears chiefly in extracellular spaces or lumens, without a convincing cellular pattern. | That compartment does not fit ACTN4's reported intracellular locations or lack of a transmembrane segment (UniProt O43707). Treat it as suspected artefact, inspect morphology and background, and require a credible cellular pattern before calling the section positive (standard IHC practice). |
| Adipocytes or cardiomyocytes stain strongly, although nearby cells can be identified reliably (HPA tissue IHC). | HPA reports ACTN4 as Not detected in those cell types (HPA tissue IHC). Check for cross-reactivity or endogenous detection activity before assigning the signal to ACTN4 (standard IHC practice). A single discordant cell type is a warning, especially because HPA cautions that its antibodies target proteins from more than one gene (HPA tissue IHC). |
| Pale chromogen covers cells, stroma, and empty regions with little cell-specific contrast. | A diffuse wash of colour is difficult to reconcile with the reported cytoplasmic tissue pattern (HPA tissue IHC). Compare a reagent control and examine whether blocking, washing, primary antibody concentration, or detection chemistry is producing background (standard IHC practice). |
| No cellular signal appears in a section containing identifiable colon endothelial cells or bladder urothelial cells (HPA tissue IHC). | Those are reported High-staining cell types, so absence of signal calls for a run check before interpreting the specimen as negative (HPA tissue IHC; standard IHC practice). Confirm tissue preservation, control performance, retrieval and detection steps, and the primary antibody's IHC validation (standard IHC practice). |
💡Expected ACTN4 appearanceCall a section positive when identifiable HPA High cell types show clear, predominantly cytoplasmic staining above local background; extracellular deposits or strong staining in HPA Not detected cell types need investigation (HPA tissue IHC; UniProt O43707; standard IHC practice).
| Tissue and cell choice | Use a reported High cell type, such as lung type II alveolar cells or placental trophoblastic cells, to judge whether a run detects the expected pattern (HPA tissue IHC). Interpret absence within a specifically identified cell type; HPA also describes cytoplasmic expression across essentially all tissue types (HPA tissue IHC). |
| Antibody evidence and specificity | HPA lists IHC as Supported for HPA001873 and HPA006035, both rabbit polyclonal antibodies (HPA antibodies). Its tissue summary reports medium staining–RNA consistency and cautions that the antibodies target proteins from more than one gene (HPA tissue IHC). An expected pattern alone therefore does not prove ACTN4-specific binding (HPA tissue IHC). |
| Compartment and nuclear signal | UniProt places ACTN4 in cytoplasm, junctions, stress fibers, perinuclear regions, and nucleus (UniProt O43707). Assess convincing nuclear staining alongside morphology and cellular cytoplasmic signal; do not automatically classify all nuclear staining as artefact, but do not infer a tissue-specific nuclear pattern from the HPA cytoplasmic IHC summary (UniProt O43707; HPA tissue IHC). |
| IF/ICC Q: What fluorescence pattern is expected? | A: HPA reports approved main locations at actin filaments and focal adhesion sites, with additional cytosol, primary cilium, and transition-zone locations (HPA subcellular ICC-IF). Its subcellular summary cautions that the antibodies target proteins from multiple genes; these IF observations do not establish an IHC retrieval setting (HPA subcellular ICC-IF). |
| Situation | Likely cause | Next action |
|---|---|---|
| A reported High cell type is unstained (HPA tissue IHC). | The run may have failed at retrieval, primary incubation, or detection (standard IHC practice). | Check an identifiable HPA High cell type in the same run, then review the antibody's IHC validation and the recorded run steps (HPA tissue IHC; standard IHC practice). |
| Only faint cytoplasmic colour appears in a reported High cell type (HPA tissue IHC). | Weak detection or limited cellular preservation can make the expected pattern hard to distinguish (standard IHC practice). | Compare run controls, inspect section morphology, and review retrieval and antibody dilution against the chosen IHC procedure (standard IHC practice). No ACTN4-specific retrieval or dilution setting is supplied here. |
| Chromogen blankets the section and obscures cell boundaries. | Background from blocking, washing, antibody concentration, or detection chemistry is possible (standard IHC practice). | Use a reagent control, review washing and blocking, and adjust the detection workflow until cellular signal can be distinguished from background (standard IHC practice). |
| An extracellular or luminal deposit is the dominant signal. | That location is inconsistent with ACTN4's listed intracellular compartments (UniProt O43707). | Inspect tissue morphology and staining deposits; repeat with appropriate run controls before scoring the deposit as positive (standard IHC practice). |
| A HPA Not detected cell type stains strongly, such as an adipocyte (HPA tissue IHC). | Cross-reactivity or endogenous detection activity is possible (standard IHC practice); HPA warns of antibodies targeting proteins from more than one gene (HPA tissue IHC). | Check a primary-omission control for detection activity and compare an independently validated antibody or orthogonal evidence if specificity is essential (standard IHC practice). |
| Nuclear staining dominates while the expected cytoplasmic pattern is unclear (HPA tissue IHC). | UniProt permits nuclear ACTN4, but the HPA tissue IHC summary describes ubiquitous cytoplasmic expression (UniProt O43707; HPA tissue IHC). | Check compartment boundaries, controls, and antibody specificity before calling the nuclear signal ACTN4; report the observed compartment separately from the expected tissue pattern (standard IHC practice). |
Experiment evidence
IHC & IF images showing ACTN4 staining
PB9974 · IHCIHC analysis of Alpha Actinin 4 using anti-Alpha Actinin 4 antibody (PB9974). Alpha Actinin 4 was detected in a paraffin-embedded section of
PB9974 · IHCIHC analysis of Alpha Actinin 4 using anti-Alpha Actinin 4 antibody (PB9974). Alpha Actinin 4 was detected in a paraffin-embedded section of
PB9974 · IFIF analysis of Alpha Actinin 4 using anti-Alpha Actinin 4 antibody (PB9974). Alpha Actinin 4 was detected in an immunocytochemical section o
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000130402-ACTN4/tissue/cerebral+cortex#imid_969608
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000130402-ACTN4/tissue/colon#imid_969534
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000130402-ACTN4/tissue/liver#imid_969543
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000130402-ACTN4/tissue/kidney#imid_969544
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA006035; green = ACTN4, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA006035; green = ACTN4, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for ACTN4 IHC & IF
🧪Run colon first and require staining in endothelial cells (High; HPA: colon endothelial cells). Use adipose tissue adipocytes as the negative reference (HPA: Not detected in adipocytes); count neighboring cells on the colon slide as internal negatives only if they are visibly unstained, since the supplied HPA row does not establish their status.
Positive control tissue: Colon (Endothelial cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show ACTN4 in A-431, PODO/SVTERT152, PODO/TERT256, ASC52telo, RPTEC/TERT1, hTERT-RPE1 (serum starved), with annotated localisation: Actin filaments (approved), Focal adhesion sites (approved) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only slide and a rabbit IgG isotype control matched to the primary antibody (caption: rabbit primary; standard IHC practice). Where available, use ACTN4 knockout material or a validated peptide-block control to check specificity, and quench endogenous peroxidase in colon sections before HRP/DAB detection (standard IHC practice; caption: HRP/DAB).
⚠️Feasibility: The selected PB9974 paraffin-section caption reports heat-mediated EDTA retrieval at pH 8.0, but does not report the fixative; a target-specific fixation window and whether retrieval is required are unreported (caption: retrieval and fixative). The supplied evidence does not compare frozen-section or IF ease with paraffin IHC; HPA reports ACTN4 at actin filaments and focal adhesions in ICC-IF, with a multiple-antibody caution (HPA: subcellular summary). For colon IHC, assess background from endogenous peroxidase before interpreting DAB staining (standard IHC practice; caption: HRP/DAB).
HPA tissue IHC evidence for ACTN4
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Supported — Medium consistency between antibody staining and RNA expression data. Caution, targets protein from more than one gene.). 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 |
|---|---|---|---|---|
| Colon | Endothelial cells | High | Protein (IHC) | HPA → |
| Liver | Cholangiocytes | High | Protein (IHC) | HPA → |
| Lung | Alveolar cells type II | High | Protein (IHC) | HPA → |
| Placenta | Trophoblastic cells | High | Protein (IHC) | HPA → |
| Urinary bladder | Urothelial cells | High | 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 → |
| Heart muscle | Cardiomyocytes | Not detected | Protein (IHC) | HPA → |
| Hippocampus | Glial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced ACTN4 IHC Tips
Troubleshoot ACTN4 staining in paraffin sections by checking retrieval, controls and cell level localisation before interpreting staining intensity.
What retrieval should I use if ACTN4 staining is weak in paraffin sections?
Start with heat mediated antigen retrieval in EDTA at pH 8.0 (datasheet PB9974). That is the retrieval used for the catalog antibody in a paraffin section of human colonic adenocarcinoma; the caption reports 2 μg/ml primary antibody incubated overnight at 4°C (datasheet PB9974). If staining remains weak, check that the section was adequately deparaffinised and that heating, cooling and washes were consistent across slides (standard IHC practice). Compare any retrieval changes on serial sections with a no primary control, because stronger colour alone does not establish ACTN4 specificity (standard IHC practice).
Could fixation explain weak or uneven ACTN4 staining?
Target specific fixation sensitivity is unknown: the selected paraffin section caption does not state its fixative (datasheet PB9974). Record the specimen fixative and processing history, then compare similarly processed sections before attributing a staining difference to ACTN4 abundance (standard IHC practice). If signal varies within a section, inspect morphology and compare well preserved areas with damaged or poorly processed areas using the same EDTA pH 8.0 retrieval (datasheet PB9974; standard IHC practice). Keep retrieval, antibody incubation and detection constant during that comparison so a processing effect is easier to distinguish from assay variation (standard IHC practice).
Should ACTN4 stain the cytoplasm, junctions or nuclei?
Expect substantial cytoplasmic staining, with possible enrichment along the actin cytoskeleton, stress fibres and cell junctions (UniProt O43707 localisation; HPA tissue IHC profile). Nuclear ACTN4 is also reported, including exclusive nuclear localisation in a limited number of cell lines, so nuclear colour is not automatically artefactual (UniProt O43707 localisation). Judge nuclear and cytoplasmic staining separately against morphology and no primary controls rather than treating a dark nucleus as proof of translocation (standard IHC practice). HPA places ACTN4 mainly at actin filaments and focal adhesion sites in its subcellular assessment, but cautions that its antibodies target proteins from multiple genes (HPA subcellular).
How can I assess whether this antibody detects the intended ACTN4 epitope?
ACTN4 has 3 listed isoforms, so determine which sequence region the catalog antibody recognises before claiming that staining represents every isoform (UniProt O43707 isoforms; standard IHC practice). The supplied caption reports staining conditions but gives no immunogen or epitope, leaving isoform coverage unresolved for PB9974 (datasheet PB9974). ACTN4 contains two calponin homology domains and two EF hands, and its listed modified residues include phosphotyrosine 31 and phosphothreonine 249 (UniProt O43707 domains and modified residues). Without epitope mapping, do not infer that retrieval or staining differences measure a particular modification or isoform; assess specificity with an independent antibody where feasible (standard IHC practice).
How should I plan IF follow-up to an ACTN4 IHC result?
Treat IF as a separate assay requiring its own antibody validation; the PB9974 evidence supplied here is for chromogenic staining of a paraffin section (datasheet PB9974; standard IF practice). Multiplex ACTN4 with an independently validated marker for the cell type being examined, such as a marker of endothelial cells when evaluating colon endothelium (HPA tissue IHC: High in colon endothelial cells; standard IF practice). Choose a fluorophore channel after checking the specimen's autofluorescence and include single stain controls to assess bleed through (standard IF practice). Because ACTN4 has no transmembrane segment and is reported in intracellular compartments, test permeabilisation for access to the antibody's epitope after fixation, documenting the IF conditions separately (UniProt O43707 topology and localisation; standard IF practice).
What should I check when the DAB signal is diffuse or widespread?
Broad cytoplasmic colour can be genuine because HPA describes ACTN4 expression across essentially all tissue types, although its tissue staining has only Supported reliability and cautions that antibodies may target more than one gene (HPA tissue IHC). First inspect a no primary section for secondary reagent or chromogen background, and verify the peroxidase block before DAB development (standard IHC practice). The PB9974 caption used 10% goat serum blocking, a peroxidase conjugated secondary and DAB detection in a paraffin section (datasheet PB9974). If colour obscures cell boundaries, compare a shorter DAB development or lower primary concentration on adjacent sections while holding retrieval constant (standard IHC practice).
How should I quantify ACTN4 staining across tissue sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring, since ACTN4 may appear in cytoplasm, junctions or nuclei (UniProt O43707 localisation; standard IHC practice). For a defined population, report the percentage of positive cells and an intensity based H score of 0–300, recording nuclear and cytoplasmic scores separately when both are present (standard IHC practice). Normalise counts to the number of evaluable cells in that population; if reporting positive cell density, use evaluable tissue area in mm² as the denominator (standard IHC practice). Apply one positivity threshold and imaging or review procedure across specimens, and report excluded damaged or necrotic regions (standard IHC practice).
How do I distinguish true ACTN4 signal from staining artefact?
Check whether colour follows identifiable cells and a plausible cytoplasmic, junctional or nuclear compartment rather than pooling over tissue edges or necrotic regions (UniProt O43707 localisation; standard IHC practice). Compare with a no primary control and a peroxidase blocked control when endogenous enzyme activity could contribute to DAB colour (standard IHC practice). Cell identity matters: HPA reports High staining in colon endothelial cells and Not detected staining in adipocytes, while describing its tissue evidence as Supported with a multiple gene antibody caution (HPA tissue IHC). Interpret a new compartment or cell pattern cautiously until it is reproducible on adjacent sections and supported by an independent specificity control (standard IHC practice).
Boster reagents
Best ACTN4 / Alpha-actinin-4 IHC Antibodies
PB9974 has IHC images from human paraffin sections and an IF image from HeLa cells (PB9974 image captions). The catalog lists human, mouse and rat reactivity (catalog: PB9974).
Real IHC data
Anti-Alpha Actinin 4/ACTN4 Antibody ®
Cat # PB9974
PB9974 is the SKU with a rendered card; its IHC captions show human colonic adenocarcinoma and prostate cancer paraffin sections (cards payload; PB9974 IHC captions). Its IF caption shows HeLa cells, while M01975 lists IHC and IF/ICC applications but has no supplied images (PB9974 IF caption; catalog: M01975).
Which to pick: For tissue IHC, choose PB9974: its captions document human paraffin sections stained at 2 μg/ml after EDTA retrieval; the fixative is unreported (PB9974 IHC captions). For IF/ICC, PB9974 has a HeLa-cell IF image at 5 μg/ml, while M01975 lists IF/ICC without a supplied image (PB9974 IF caption; catalog: M01975). For cross-species work, both list human, mouse and rat reactivity; consider M01975 if a monoclonal antibody is preferred, but the supplied tissue IHC images are human only (catalog: PB9974, M01975; PB9974 IHC captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry O43707 (ACTN4_HUMAN, Alpha-actinin-4).
- Human Protein Atlas. ACTN4 tissue IHC expression (reliability: Supported).
- Human Protein Atlas. ACTN4 subcellular location (ICC-IF): Mainly localized to the actin filaments and focal adhesion sites. In addition localized to the cytosol, primary cilium and primary cilium transition zone. Caution: Based on antibodies targeting proteins from multiple genes..
- Human Protein Atlas. ACTN4 antibody validation summary (2 antibodies).
- Alpha-actinin 4 and tumorigenesis of hepatocellular carcinoma. Translational cancer research 2019 — PMC8798106.
- Copy number gain of ACTN4 is associated with poor prognosis in patients with upper urinary tract urothelial carcinoma. Cancer science 2023 — PMC10394141.
- Prognostic Significance of Actinin-4 Protein Expression and Gene Amplification in Endometrial Carcinoma. The journal of obstetrics and gynaecology research 2025 — PMC12611447.
- ACTN4 is associated with the malignant potential of thymic epithelial tumors through the β-catenin/Slug pathway. Cancer science 2024 — PMC11531964.
- PubMed PMID:10656685 — UniProt-cited evidence.
- PubMed PMID:22567897 — UniProt-cited evidence.
- PubMed PMID:15057824 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for ACTN4 (Alpha-actinin-4). Expected localisation Predominantly cytoplasmic in tissue (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet PB9974). Validated IHC antibody: PB9974 at 2-5μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Colon (Endothelial cells); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human colonic adenocarcinoma tissue; fixative not specified (datasheet PB9974); 2. Fixation: Image fixative and duration unreported (datasheet PB9974); 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 PB9974); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet PB9974); 8. Primary antibody: Rabbit anti-ACTN4, 2-5μg/ml (datasheet PB9974); 9. Primary incubation: Overnight at 4 °C (datasheet PB9974). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific); Caveat: Antibody staining may reflect more than one gene (HPA tissue IHC); Isoform / epitope: 3 isoforms; epitope coverage is unspecified (UniProt); Regulation: Blood-vessel-enhanced RNA expression (HPA tissue RNA). Evidence: UniProt O43707, HPA tissue IHC · Supported, HPA ICC-IF · Mainly localized to the actin filaments and focal adhesion sites. In…, Datasheet PB9974, PMC8798106, PMC10394141. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/ACTN4/immunohistochemistry-ihc-guide.