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ACTB / Actin, cytoplasmic 1 · IHC design guide
Design Immunohistochemistry for ACTB
Plan ACTB chromogenic IHC in paraffin sections using the catalog antibody at 1:25 (datasheet: M01263-3). Assess myoepithelial staining (HPA tissue IHC) and account for possible detection of products from more than one gene (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
ACTB 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 | Cytoskeletal and nuclear locations are annotated; the tissue compartment is unreported (UniProt; HPA tissue IHC) | |
| Staining pattern | High myoepithelial staining across tissues; compartment unreported (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6 HIER, heat-mediated (datasheet M01263-3) | |
| Positive control | Appendix+4 more · see all | |
| Negative control | Adrenal gland+4 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Formaldehyde-fixed paraffin sections are shown (selected-SKU IHC image M01263-3); Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image M01263-3) | |
| Caveat | The antibody may detect products of more than one gene (HPA tissue IHC) | |
| Regulation | Low tissue specificity; an intensity regulator is unreported (HPA tissue IHC) | |
| Isoform / epitope | No annotated isoforms; N-terminal processing may affect epitopes (UniProt) |
Section 1
Recommended ACTB IHC & IF Protocols
The catalog antibody protocol and the published ACTB IHC protocol below both use citrate pH 6 retrieval (datasheet: citrate pH 6; PMC12446288: citrate pH 6).
| Sample | Formaldehyde-fixed, paraffin-embedded human heart tissue (datasheet M01263-3) |
| Fixation | Image formalin-fixed; duration unreported (datasheet M01263-3); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: Citrate pH 6 (datasheet M01263-3); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% normal serum of the secondary host, 30 min, room temperature (standard) |
| Primary antibody | Mouse monoclonal (clone 137CT26.1.1) anti-ACTB, 1:25 (datasheet M01263-3) |
| Primary incubation | 1 hours at 37°C (datasheet M01263-3) |
| Detection | HRP-polymer secondary, DAB chromogen 5–10 min (standard) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | ACTB-positive staining in enterocytes - Microvilli of appendix (HPA tissue IHC: High). HPA tissue profile: High myoepithelial expression in all tissues. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated citrate pH 6 retrieval for the catalog antibody (datasheet: citrate pH 6); the published protocol uses microwave retrieval at the same pH (PMC12446288).
Section 2
What Is the Expected ACTB Staining Pattern?
ACTB localises to the cytoplasmic cytoskeleton and can also occur in the nucleus; it has no transmembrane segment (UniProt P60709). Expect high myoepithelial staining across tissues and high enterocyte microvillar staining in appendix, colon and duodenum (HPA: tissue IHC). HPA rates the tissue pattern Supported, with medium consistency and a caution that staining may detect products of more than one gene (HPA: reliability).
| Strong breast myoepithelial staining (HPA: High). | Matches the reported ACTB tissue pattern (HPA: breast myoepithelial cells High). |
| Extracellular-only chromogen. | Inconsistent with intracellular ACTB; suspect artefact (UniProt P60709 topology). |
| Strong bronchial respiratory epithelial staining. | Unexpected (HPA: Not detected); consider cross-reactivity or endogenous enzyme activity (standard IHC practice). |
| Diffuse chromogen over cells and empty areas. | Suggests nonspecific background or detection artefact (standard IHC practice). |
| No signal in breast myoepithelial cells. | Unexpected in a reported positive cell type (HPA: breast myoepithelial cells High). |
💡Expected ACTB appearanceStrong intracellular myoepithelial staining is expected (HPA: High; UniProt P60709); extracellular-only chromogen is suspect (UniProt P60709 topology).
| Antibody specificity (HPA: reliability) | IHC is Supported, but staining may detect products of more than one gene (HPA: caution). |
| Nuclear localisation (UniProt P60709) | Nuclear signal can be plausible alongside cytoskeletal staining (UniProt P60709). |
| N-terminal processing (UniProt P60709) | Residues 1–375 and 2–375 are annotated; check epitope position if known (UniProt P60709). |
| Cell-type variation (HPA: tissue IHC) | High breast myoepithelial and undetected bronchial epithelial staining are reported (HPA: tissue IHC). |
| Situation | Likely cause | Next action |
|---|---|---|
| No breast myoepithelial signal (HPA: High). | Assay failure is possible (standard IHC practice). | Check positive control, retrieval, antibody dilution and detection (standard IHC practice). |
| Diffuse slide background. | Inadequate blocking or washing is possible (standard IHC practice). | Review blocking, washes and detection controls (standard IHC practice). |
| Extracellular-only chromogen. | Precipitate or detection artefact is possible (standard IHC practice). | Inspect reagent cleanliness and a no-primary control (standard IHC practice). |
| Strong bronchial respiratory epithelial signal (HPA: Not detected). | Cross-reactivity or endogenous enzyme activity is possible (HPA: caution; standard IHC practice). | Compare an independent antibody and a no-primary control (standard IHC practice). |
| Nuclear-only breast myoepithelial signal. | The expected cytoskeletal component is absent (UniProt P60709). | Review controls and intracellular staining distribution (standard IHC practice). |
| IF/ICC: is a nuclear signal plausible? | ACTB can be nuclear; HPA provides no ICC-IF images (UniProt P60709; HPA: subcellular). | Interpret with IF controls and the separate IF/ICC guide (standard IF practice). |
Experiment evidence
IHC & IF images showing ACTB staining
M01263-3 · IHCM01263-3 staining ACTB in human heart tissue sections by Immunohistochemistry (IHC-P -paraformaldehyde-fixed, paraffin-embedded sections). T
M01263-3 · IHCImmunohistochemical analysis of paraffin-embedded H.skeletal muscle section using Beta-Actin Antibody. M01263-3 was diluted at 1:25 dilution
M01263-3 · IFConfocal immunofluorescent analysis of ACTB Antibody with Hela cell followed by Alexa Fluor® 488-conjugated goat anti-mouse lgG (green). DAP
M01263 · IHCImmunohistochemical analysis of paraffin-embedded (1) Human heart; (2) Mouse testis; (3) Human bladder cancer; (4) Mouse heart, using beta A
M01263 · IFImmunofluorescent analysis using the Antibody at 1:50 dilution.
M01263-4 · IHCImmunohistochemical analysis of paraffin-embedded human heart, using beta Actin Mouse Monoclonal Antibody.
M01263-4 · IFImmunofluorescent analysis of HepG2 cells, using beta Actin mAb.
M01263-8 · IHCHuman colon was stained with anti-β Actin rabbit antibody
A01263-1 · IFImmunocytochemistry of beta-actin in HeLa cells with beta-actin antibody at 10 μg/mL.
A01263-2 · IFImmunocytochemistry of Actin in HeLa cells with Actin antibody at 1 μg/mL.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000075624-ACTB/tissue/cerebral+cortex#imid_12522665
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000075624-ACTB/tissue/colon#imid_12526415
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000075624-ACTB/tissue/liver#imid_12526408
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000075624-ACTB/tissue/kidney#imid_12526425
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for ACTB IHC & IF
🧪Run colon first: enterocyte microvilli should stain strongly (HPA: High in colon enterocyte microvilli). Use adrenal gland glandular cells as the negative tissue (HPA: Not detected in adrenal gland glandular cells); no ACTB-negative cell population is established within the colon slide, so unstained cells there are only background comparators, not validated internal negatives (HPA: colon enterocyte microvilli High).
Positive control tissue: Appendix (Enterocytes - Microvilli, HPA High)
Negative control tissue: Adrenal gland (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA carries no ICC-IF cell line for ACTB; derive a cell-line control from the positive tissue's cell type (Enterocytes - Microvilli) and confirm it by RNA or western blot first.
Technical controls: Include no-primary (secondary-only) and host-species-matched control immunoglobulin; for a monoclonal primary, match its isotype, and for a polyclonal primary, use nonimmune IgG from the same host species (standard IHC practice). A validated ACTB-knockout specimen can provide a biological negative (standard IHC practice). Block endogenous peroxidase for chromogenic detection and check endogenous biotin if using the caption’s biotinylated secondary antibody (selected-SKU IHC caption; standard IHC practice).
⚠️Feasibility: The selected-SKU caption supports heat-mediated citrate retrieval at pH 6 for paraffin-section IHC, but does not establish that retrieval is required for every antibody or specimen (selected-SKU IHC caption). A target-specific fixation window or fixation effect is unreported, and the selected-SKU fixative is unreported in the supplied fixative field (selected-SKU caption metadata: fixative not stated); no matched frozen-section or IF evidence establishes that either is easier (supplied application evidence). In colon, assess apical microvillus staining against slide morphology and the no-primary control so edge-associated chromogen is not mistaken for specific staining (HPA: High in colon enterocyte microvilli; standard IHC practice).
HPA tissue IHC evidence for ACTB
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 |
|---|---|---|---|---|
| Appendix | Enterocytes - Microvilli | High | Protein (IHC) | HPA → |
| Breast | Myoepithelial cells | High | Protein (IHC) | HPA → |
| Colon | Enterocytes - Microvilli | High | Protein (IHC) | HPA → |
| Duodenum | Enterocytes - Microvilli | High | Protein (IHC) | HPA → |
| Epididymis | Glandular cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adrenal gland | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | 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 | Glial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced ACTB IHC Tips
Use the catalog antibody’s tissue staining conditions as a starting point, then assess ACTB signal by compartment, cell type, and controls.
How should I retrieve ACTB in paraffin sections when staining is weak?
Start with heat-mediated citrate buffer at pH 6 for ACTB antigen retrieval (datasheet M01263-3). The selected tissue image used this retrieval before a 1:25 primary antibody incubation for 1 hour at 37°C (caption M01263-3). If staining is weak, check section adhesion, heating consistency, and reagent performance before comparing a second retrieval condition on adjacent sections (standard IHC practice). Keep the antibody concentration and detection system constant during that comparison so the retrieval effect can be judged (standard IHC practice). Examine cytoplasmic detail and tissue preservation alongside signal intensity, because excessive retrieval can damage morphology (standard IHC practice).
Could fixation explain weak or uneven ACTB staining?
ACTB-specific sensitivity to fixation is unknown from the supplied evidence, so an uneven stain cannot be assigned to a particular fixative or fixation time. For paraffin IHC, document the actual fixative, fixation duration, tissue thickness, and processing history for each specimen before comparing results (standard IHC practice). Compare sections processed together and include a known staining control in the same run to separate specimen handling from assay failure (standard IHC practice). If staining differs across a block, inspect tissue preservation and the section edge before adjusting antibody concentration (standard IHC practice). Record any fixation change as an experimental variable, not an established ACTB-specific requirement.
Where should ACTB staining appear, and when is nuclear signal plausible?
Assess cytoplasmic and cytoskeletal staining first, because ACTB is annotated in the cytoplasm and cytoskeleton (UniProt P60709 localisation). Nuclear ACTB is also annotated, including roles for nuclear actin, so nuclear staining alone is not automatically artefactual (UniProt P60709 localisation and function). Evaluate nuclear signal against matched controls, morphology, and the surrounding cytoplasmic pattern before interpreting it (standard IHC practice). ACTB can occur in cytoplasmic mRNP granules, but routine chromogenic IHC cannot identify those complexes solely from a punctate pattern (UniProt P60709 localisation; standard IHC practice). Record compartment-specific staining separately when comparing specimens (standard IHC practice).
Can this stain distinguish ACTB from other actins or modified ACTB?
Do not infer antibody specificity from the gene name alone: the supplied record lists 0 isoforms but does not define this antibody’s epitope (UniProt P60709 isoforms; supplied antibody evidence). ACTB has an N-terminally processed chain beginning at residue 2 and annotated modifications including N-terminal acetylation, methionine oxidation, and histidine methylation (UniProt P60709 processing and modified residues). Whether any of these features affects this antibody’s tissue staining is unknown without epitope or validation data. Compare an appropriate specificity control with the same retrieval and detection conditions before attributing a staining change to ACTB (standard IHC practice). HPA also cautions that its tissue staining targets protein from more than one gene (HPA tissue IHC reliability description).
How can I compare ACTB immunofluorescence with the chromogenic tissue result?
Treat IF/ICC as a separate validation experiment; the supplied 1:25 incubation and citrate pH 6 retrieval describe tissue IHC, not IF/ICC conditions (caption M01263-3). Multiplex ACTB with an independently validated marker for the cell type being examined, and use single-stain controls to check channel bleed-through (standard IF practice). Choose a fluorophore whose emission can be distinguished from the specimen’s autofluorescence, using an unstained section to assess that background (standard IF practice). ACTB has no transmembrane segment and is annotated in cytoplasm, cytoskeleton, and nucleus; select permeabilisation that permits access to the compartment of interest without assuming a mapped antibody epitope (UniProt P60709 topology and localisation; standard IF practice).
What should I check when ACTB staining is diffuse or unexpectedly strong?
First compare a no-primary control with the stained section to assess detection-system background (standard IHC practice). For chromogenic detection, check endogenous peroxidase blocking and inspect pigment or precipitate before interpreting diffuse brown signal as ACTB (standard IHC practice). The selected image used 3% BSA for 0.5 hour at room temperature and a biotinylated secondary reagent, so assess blocking and biotin-related background when reproducing that setup (caption M01263-3; standard IHC practice). Titrate the primary antibody around the documented 1:25 condition while holding retrieval and detection constant (caption M01263-3; standard IHC practice). Preserve counterstain and exposure conditions across comparisons (standard IHC practice).
How should I quantify ACTB across paraffin tissue sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring, then use either percentage of positive cells or an H-score based on staining intensity and percentage (standard IHC practice). An H-score spans 0–300 when intensity categories 0–3 are weighted by their cell percentages (standard IHC practice). Normalise counts to the number of evaluable cells, or stained area to the measured tissue area in mm², and exclude folds, necrosis, and missing tissue by a prespecified rule (standard IHC practice). Report cytoplasmic and nuclear scores separately because both localisations are annotated for ACTB (UniProt P60709 localisation). Use matched processing and detection settings across specimens (standard IHC practice).
How can I distinguish genuine ACTB staining from a tissue artefact?
Look for reproducible cellular staining with preserved morphology and a pattern compatible with ACTB’s cytoplasmic, cytoskeletal, or nuclear localisation (UniProt P60709 localisation; standard IHC practice). Check whether the stained cells match the population being scored: HPA reports high myoepithelial expression and high staining in several listed cell populations, but cautions that its antibody targets protein from more than one gene (HPA tissue IHC profile and reliability description). Treat isolated section-edge staining, necrotic deposits, and signal in the no-primary control as possible artefacts (standard IHC practice). Check endogenous enzyme activity when using chromogenic detection (standard IHC practice). Confirm unexpected patterns with an independent specificity control before assigning them to ACTB (standard IHC practice).
Boster reagents
Best ACTB / Actin, cytoplasmic 1 IHC Antibodies
Anti-ACTB antibodies have IHC images from human and mouse tissues and IF/ICC images from HeLa cells; catalog reactivity spans human, mouse, rat, chicken, pig and rabbit (catalog image captions and reactivity).
Real IHC data
Anti-Beta-Actin Antibody
Cat # M01263-3
Real IHC data
Anti-beta Actin ACTB Rabbit Monoclonal Antibody
Cat # M01263
Real IHC data
Anti-beta Actin Mouse Monoclonal Antibody
Cat # M01263-4
Real IHC data
Anti-β Actin Rabbit Monoclonal Antibody
Cat # M01263-8
Real IF data
Anti-Beta-actin ACTB Antibody
Cat # A01263-1
Real IF data
Anti-beta-Actin ACTB Antibody
Cat # A01263-2
M01263-3 shows human heart IHC-P (M01263-3 image caption); M01263 shows human heart, human bladder cancer, mouse testis and mouse heart IHC (M01263 image caption); M01263-4 shows human heart IHC (M01263-4 image caption); M01263-8 shows human colon staining (M01263-8 image caption). A01263-1 shows HeLa ICC and IF (A01263-1 image captions), while A01263-2 shows HeLa ICC and IF (A01263-2 image captions).
Which to pick: For paraffin-section IHC, choose M01263-3: its human heart image specifies formaldehyde fixation, citrate retrieval at pH 6 and a 1:25 primary dilution (M01263-3 image caption). For IF/ICC, A01263-1 lists both applications and has HeLa images for each (A01263-1 applications and image captions). For broader listed species reactivity, M01263-4 covers chicken, human, mouse, pig, rabbit and rat; its IHC image shows paraffin-embedded human heart, but the fixative is unreported (M01263-4 catalog reactivity and IHC image caption).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P60709 (ACTB_HUMAN, Actin, cytoplasmic 1).
- Human Protein Atlas. ACTB tissue IHC expression (reliability: Supported).
- Human Protein Atlas. ACTB subcellular location (ICC-IF): Highest expression in PODO/TERT256: 12477.8 nTPM.
- Human Protein Atlas. ACTB antibody validation summary (3 antibodies).
- Differential tumor protein expression at follicular lymphoma diagnosis reveals dysregulation of key molecular pathways associated with histological transformation. Scientific reports 2024 — PMC11612490.
- ACTB may serve as a predictive marker for the efficacy of lenvatinib in patients with HBV-related early-stage hepatocellular carcinoma following partial hepatectomy: a retrospective cohort study. Journal of gastrointestinal oncology 2023 — PMC10772687.
- Exploring heterogeneous expression of beta-actin (ACTB) in bladder cancer by producing a monoclonal antibody 6D6. BMC urology 2024 — PMC11167769.
- Altered actin isoforms expression and enhanced airway responsiveness in asthma: the crucial role of β-cytoplasmic actin. Frontiers in physiology 2025 — PMC12446288.
- PubMed PMID:6322116 — UniProt-cited evidence.
- PubMed PMID:2994062 — UniProt-cited evidence.
- PubMed PMID:1734024 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for ACTB (Actin, cytoplasmic 1). Expected localisation Cytoskeletal and nuclear locations are annotated; the tissue compartment is unreported (UniProt; HPA tissue IHC). Antigen retrieval: Citrate pH 6 HIER, heat-mediated (datasheet M01263-3). Validated IHC antibody: M01263-3 at 1:25 (the product's own IHC validation image is shown). Controls (HPA): positive Appendix (Enterocytes - Microvilli); negative Adrenal gland. IHC-P workflow on paraffin sections. Protocol steps: 1. Sample: Formaldehyde-fixed, paraffin-embedded human heart tissue (datasheet M01263-3); 2. Fixation: Image formalin-fixed; duration unreported (datasheet M01263-3); 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: Citrate pH 6 (datasheet M01263-3); 20 min, 95–100 °C (standard); 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: Mouse monoclonal (clone 137CT26.1.1) anti-ACTB, 1:25 (datasheet M01263-3); 9. Primary incubation: 1 hours at 37°C (datasheet M01263-3). Watch for: Fixation: Formaldehyde-fixed paraffin sections are shown (selected-SKU IHC image M01263-3); Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image M01263-3); Caveat: The antibody may detect products of more than one gene (HPA tissue IHC); Isoform / epitope: No annotated isoforms; N-terminal processing may affect epitopes (UniProt); Regulation: Low tissue specificity; an intensity regulator is unreported (HPA tissue IHC). Evidence: UniProt P60709, HPA tissue IHC · Supported, HPA cell-line RNA · Highest expression in PODO/TERT256: 12477.8 nTPM, Datasheet M01263-3, PMC12446288. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/ACTB/immunohistochemistry-ihc-guide.