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ACTN4 / Alpha-actinin-4 · IHC design guide
Design Immunohistochemistry for ACTN4
Plan ACTN4 chromogenic IHC on paraffin sections using the catalog antibody’s documented conditions. Compare cytoplasmic staining with HPA tissue patterns and include appropriate controls when interpreting cell-specific signal.
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 | Cytoplasmic tissue staining (HPA tissue IHC) | |
| Staining pattern | Cytoplasmic signal 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). Selected-image fixative and duration unreported (datasheet PB9974); verify before use. | |
| Caveat | Staining may reflect more than one gene product (HPA tissue IHC) | |
| Regulation | Widely expressed; no inducer specified (UniProt) | |
| Isoform / epitope | 3 isoforms; verify the antibody epitope across them (UniProt) |
Section 1
Recommended ACTN4 IHC & IF Protocols
The catalog antibody protocol uses EDTA retrieval (datasheet: PB9974); the published ACTN4 IHC protocols below use citrate retrieval (PMC8798106; 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). If adapting a published protocol, follow its reported citrate conditions (PMC8798106; PMC10394141).
Section 2
What Is the Expected ACTN4 Staining Pattern?
ACTN4 is a widely expressed, non-transmembrane protein that localises mainly to the cytoplasm, cytoskeleton and cell junctions; nuclear localisation is also reported (UniProt O43707). For paraffin-section IHC, expect cytoplasmic staining across many tissues, with high staining reported in colon endothelial cells, liver cholangiocytes and lung type II alveolar cells (HPA: tissue IHC). HPA rates the 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).
| Clear cytoplasmic staining in colon endothelial cells or liver cholangiocytes, with cell outlines remaining interpretable. | This fits reported high staining in those cell types and the expected cytoplasmic distribution (HPA: tissue IHC; UniProt O43707). Judge the intended cells separately from adjacent cells: a positive tissue section does not mean every cell in it must have the same intensity (HPA: tissue IHC). |
| Signal appears only at an extracellular surface or as a continuous outline around every cell, without discernible cytoplasm. | That is inconsistent with ACTN4's non-transmembrane topology and reported intracellular locations (UniProt O43707). Treat the compartment mismatch as a possible artefact; review the chromogenic detection and staining controls before scoring the section as positive (standard IHC practice). Nuclear signal alone needs separate assessment because nuclear localisation is reported (UniProt O43707). |
| Strong staining is assigned to adipocytes, cardiomyocytes or skeletal myocytes. | HPA reports ACTN4 as not detected in those specific cell types, despite its broad tissue distribution (HPA: tissue IHC). Check cell identity, nearby positive cells and control sections; cross-reactivity or endogenous detection activity can produce misleading signal (HPA: multi-gene antibody caution; standard IHC practice). |
| Brown colour is diffuse across tissue spaces, edges or the whole section, obscuring individual cells. | A diffuse deposit cannot establish the intracellular pattern expected for ACTN4 (UniProt O43707). Assess a no-primary control and the detection reagents, then optimise blocking, washes and chromogen development as needed (standard IHC practice). Avoid scoring intensity where cellular boundaries cannot be resolved (standard IHC practice). |
| No stain appears in a section containing an identified high-staining cell population. | Absent signal in colon endothelial cells, liver cholangiocytes or lung type II alveolar cells conflicts with the reported high IHC levels (HPA: tissue IHC). First verify that the relevant cells are present, then check the antibody's IHC-P instructions, detection run and positive control (standard IHC practice). The HPA level alone does not identify a failure mechanism. |
💡Expected ACTN4 appearanceCall a section positive when identifiable cells show predominantly cytoplasmic staining, including strong signal in an HPA high-staining population; diffuse extracellular colour or uniform outlines without cellular detail are suspect (UniProt O43707; HPA: tissue IHC; standard IHC practice).
| Cell and tissue selection | ACTN4 is widely expressed (UniProt O43707), while HPA reports high staining in specified cells and no detection in others within named tissues (HPA: tissue IHC). Select and score controls by cell type, not by tissue name alone (standard IHC practice). |
| Compartment and topology | ACTN4 has no transmembrane segment; UniProt lists cytoplasmic, cytoskeletal, junctional, perinuclear and nuclear locations (UniProt O43707). A nuclear component can be plausible, but an exclusively nuclear IHC pattern should be interpreted in its cell context rather than assumed to be the routine tissue pattern (UniProt O43707; HPA: tissue IHC). |
| Antibody evidence and scope | HPA lists IHC as Supported for HPA001873 and HPA006035, and cautions that its staining evidence involves antibodies targeting proteins from more than one gene (HPA: antibody validation; HPA: tissue IHC). Agreement with an expected pattern supports interpretation but does not, by itself, prove ACTN4-specific binding. |
| Can IF/ICC findings guide IHC interpretation? | Yes, for compartment context: HPA reports actin filaments and focal adhesion sites as approved main ICC-IF locations, with cytosol among approved additional locations (HPA: subcellular ICC-IF). Its multi-gene antibody caution applies; fluorescence morphology does not set a chromogenic IHC-P protocol or tissue staining threshold (HPA: subcellular ICC-IF; standard IHC practice). |
| Situation | Likely cause | Next action |
|---|---|---|
| The expected high-staining cells are absent from the field. | A control chosen by organ name may miss the cell population on which the reported level is based (HPA: tissue IHC). | Locate colon endothelial cells, liver cholangiocytes or lung type II alveolar cells before judging the run; choose another supplied high-staining cell population if needed (HPA: tissue IHC). |
| Identified high-staining cells are present but show no signal. | The result conflicts with their reported HPA level; that observation alone does not establish whether antibody, retrieval or detection failed (HPA: tissue IHC). | Review the catalog antibody's IHC-P instructions and the run's positive control, retrieval, primary-antibody step and detection steps (standard IHC practice). Do not infer ACTN4-specific fixation sensitivity from the supplied evidence. |
| The whole slide has weak, diffuse brown background. | Diffuse colour may reflect nonspecific detection, insufficient washing or overdevelopment (standard IHC practice); it is not a resolved ACTN4 compartment pattern (UniProt O43707). | Compare a no-primary control, then check blocking, washes and chromogen development (standard IHC practice). Score only identifiable cells after background is controlled. |
| Reported negative cell types appear strongly positive. | HPA reports no detection in adipocytes, cardiomyocytes and skeletal myocytes, and warns that its antibodies can target proteins from more than one gene (HPA: tissue IHC). Endogenous detection activity is another general IHC possibility (standard IHC practice). | Confirm the cell type and examine no-primary and detection controls before assigning ACTN4 positivity (standard IHC practice). |
| Staining forms a uniform surface rim with little cytoplasmic detail. | A purely surface pattern does not fit ACTN4's non-transmembrane topology or reported intracellular distribution (UniProt O43707). | Inspect tissue morphology and controls, and review chromogen development and detection conditions before interpreting the rim (standard IHC practice). |
| Nuclear staining dominates while the expected cytoplasmic pattern is unclear. | UniProt permits nuclear localisation, including exclusive nuclear localisation in a limited number of cell lines; HPA describes tissue IHC as ubiquitously cytoplasmic (UniProt O43707; HPA: tissue IHC). | Record nuclear and cytoplasmic signal separately, verify the cell population and compare staining controls. Do not score nuclear signal as an automatic failure or as sufficient proof of ACTN4 specificity (standard IHC practice; HPA: multi-gene antibody caution). |
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: endothelial cells should show High ACTN4 staining (HPA: Colon, endothelial cells, High). Use adipose tissue as the negative comparator because adipocytes are Not detected (HPA: Adipose tissue, adipocytes, Not detected); on the colon slide, treat unstained cells as a background reference without assuming every non-endothelial cell is ACTN4-negative (UniProt O43707: widely expressed).
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, a concentration-matched nonimmune rabbit IgG isotype control, and a matched ACTN4 knockout biological control (selected PB9974 IHC caption: rabbit primary; standard IHC control practice). Quench endogenous peroxidase and check vascular background in the colon section before interpreting DAB staining (HPA: Colon, endothelial cells, High; standard chromogenic IHC practice).
⚠️Feasibility: No target-specific fixation window or fixation effect is reported in the supplied evidence; the fixative in the selected PB9974 paraffin-section caption is unreported (selected PB9974 IHC caption). The caption uses heat retrieval in EDTA at pH 8.0, but does not establish that retrieval is required under every processing condition (selected PB9974 IHC caption). The supplied evidence does not establish whether frozen sections or IF are easier; in colon, interpret staining by cell type because the HPA positive call is for endothelial cells (HPA: Colon, endothelial cells, High).
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, compartment, controls and scoring before interpreting chromogenic signal.
Which retrieval condition should I use when ACTN4 staining is weak in paraffin sections?
Start with heat mediated retrieval in EDTA at pH 8.0 for paraffin sections (datasheet PB9974). The documented PB9974 example then used 10% goat serum blocking and 2 μg/ml primary antibody overnight at 4°C, so check those steps before changing retrieval (datasheet PB9974). Compare a test section with the documented condition and assess signal in expected cytoplasmic structures, including actin associated regions, alongside background (UniProt O43707 localisation; standard IHC practice). If staining remains weak, test another retrieval buffer as a fallback on matched sections, keeping detection and exposure conditions constant; the supplied evidence does not establish a second ACTN4 retrieval condition (standard IHC practice; datasheet PB9974).
Could fixation explain weak or uneven ACTN4 staining?
The PB9974 tissue caption identifies a paraffin section but does not state its fixative, so target specific ACTN4 fixation sensitivity is unknown (datasheet PB9974). Record the fixative, fixation duration and processing history for each specimen, then compare sections processed together before attributing a staining difference to biology (standard IHC practice). Keep the documented EDTA pH 8.0 retrieval and 2 μg/ml primary antibody conditions constant during that comparison (datasheet PB9974). Uneven staining across similarly processed regions warrants inspection for section damage and variable processing, while a matched positive control helps identify a run level failure (standard IHC practice).
Should ACTN4 stain the nucleus, cytoplasm or cell borders?
Expect predominantly cytoplasmic staining in tissue sections, while ACTN4 can also occupy the nucleus, cell junctions, stress fibers and perinuclear region (HPA tissue IHC; UniProt O43707 localisation). Its actin cross linking role supports assessing signal in cytoskeletal and junction associated regions rather than scoring every brown deposit as equivalent (UniProt O43707 function; standard IHC practice). Nuclear staining can be biologically plausible, but UniProt describes exclusive nuclear localisation only in a limited number of cell lines; it does not establish that pattern for an arbitrary tissue section (UniProt O43707 localisation). Compare nuclear and cytoplasmic compartments separately against the counterstain and controls before calling a localisation shift (standard IHC practice).
Can this stain distinguish ACTN4 isoforms or reveal epitope masking?
ACTN4 has 3 listed isoforms and calponin homology domains at residues 50–154 and 163–269, but the supplied PB9974 caption does not identify the antibody epitope (UniProt O43707 isoforms and domains; datasheet PB9974). Do not assign a staining pattern to one isoform without epitope mapping or an independently validated isoform specific reagent (standard IHC practice). ACTN4 also has recorded modified residues, including phosphorylation and acetylation sites, yet their effects on this antibody’s tissue staining are unreported (UniProt O43707 modified residues; datasheet PB9974). If a specimen stains unexpectedly, compare matched retrieval conditions and an appropriate independent ACTN4 assay before inferring epitope loss (standard IHC practice).
How should I check an ACTN4 IHC pattern by multiplex IF?
Use IF as a separate validation experiment and pair ACTN4 with a marker for the cell population being assessed, such as an endothelial marker when examining colon endothelial cells (HPA tissue IHC: high in colon endothelial cells; standard IF practice). Choose fluorophores after checking the specimen’s autofluorescence and reserve a well separated channel for ACTN4 when background is strongest in another channel (standard IF practice). ACTN4 has no transmembrane segment and is reported in cytoplasm, cytoskeleton and nucleus, so permeabilisation should allow access to intracellular epitopes while preserving structure (UniProt O43707 topology and localisation; standard IF practice). Optimise IF fixation, permeabilisation and antibody concentration independently; the paraffin IHC caption supplies no IF settings (datasheet PB9974).
How can I reduce diffuse DAB background without losing ACTN4 signal?
First inspect a no primary control and confirm that the peroxidase block, washes and DAB development are working as intended (standard IHC practice). The documented PB9974 example used 10% goat serum blocking, 2 μg/ml primary antibody overnight at 4°C, and a peroxidase conjugated secondary for 30 minutes at 37°C (datasheet PB9974). If background persists, compare a lower primary concentration or shorter chromogen development on matched sections while retaining an expected positive area (standard IHC practice). Because HPA describes widespread cytoplasmic ACTN4 staining, diffuse tissue signal alone cannot establish nonspecific binding; judge its compartment and control pattern together (HPA tissue IHC; standard IHC practice).
What should I measure when ACTN4 staining varies across cells? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring, because ACTN4 is reported in cytoplasm and sometimes nucleus or junction associated structures (UniProt O43707 localisation; standard IHC practice). For a defined population, record the percentage of positive cells and an H-score based on intensity categories, then report nuclear and cytoplasmic scores separately if both are present (standard IHC practice). Normalise counts to the number of evaluable cells, or stained cell density to tissue area in mm², using the same threshold across matched sections (standard IHC practice). Exclude folds, necrotic regions and section edges from the analysis mask, and retain representative images with the scoring rules (standard IHC practice).
How do I distinguish credible ACTN4 staining from tissue artefact?
A credible pattern should have interpretable intracellular signal and fit the cell population being scored: HPA reports high staining in colon endothelial cells and a broadly cytoplasmic tissue profile (HPA tissue IHC). ACTN4 may also localise to junctions or nuclei, so a nuclear signal needs compartment specific review rather than automatic rejection (UniProt O43707 localisation; standard IHC practice). Treat staining confined to section edges, damaged or necrotic areas, or a no primary control as suspect; check endogenous peroxidase activity when DAB persists without primary antibody (standard IHC practice). HPA rates tissue evidence as supported but cautions that its antibodies target proteins from more than one gene, so confirm a consequential ACTN4 specific interpretation independently (HPA tissue IHC).
Boster reagents
Best ACTN4 / Alpha-actinin-4 IHC Antibodies
PB9974 has IHC data from human paraffin sections and IF/ICC data from Hela cells; its listed reactivity includes human, mouse and rat (PB9974 image captions and catalog).
Real IHC data
Anti-Alpha Actinin 4/ACTN4 Antibody ®
Cat # PB9974
PB9974 is the card that will render, with IHC images from human colonic adenocarcinoma and prostate cancer paraffin sections (PB9974 IHC captions). Its IF image shows Hela cells (PB9974 IF caption).
Which to pick: Choose PB9974 for tissue IHC: its image captions document human paraffin sections, 2 μg/ml antibody and EDTA retrieval at pH 8.0; the fixative is unreported (PB9974 IHC captions). Choose PB9974 for IF/ICC when an imaged example is useful: its caption documents Hela cells at 5 μg/ml (PB9974 IF caption). For cross-species planning, PB9974 and the monoclonal M01975 both list human, mouse and rat reactivity, but the supplied tissue IHC images document human samples only (catalog reactivity; 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 Cytoplasmic tissue staining (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). Selected-image fixative and duration unreported (datasheet PB9974); verify before use.; Caveat: Staining may reflect more than one gene product (HPA tissue IHC); Isoform / epitope: 3 isoforms; verify the antibody epitope across them (UniProt); Regulation: Widely expressed; no inducer specified (UniProt). 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.