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ACTG2 / Actin, gamma-enteric smooth muscle · IHC design guide
Design Immunohistochemistry for ACTG2
Plan ACTG2 chromogenic IHC in paraffin sections using the reported cytoplasmic tissue pattern and intestinal smooth muscle expression (HPA tissue IHC; UniProt). Compare staining with appropriate controls because the HPA cautions that its antibodies target proteins from more than one gene (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
ACTG2 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 myoepithelial cells (HPA tissue IHC) | |
| Staining pattern | Cytoplasmic staining in myoepithelial cells (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen) | |
| Positive control | Appendix+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); Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A05066-1) | |
| Caveat | Staining may reflect proteins from more than one gene (HPA tissue IHC) | |
| Regulation | Enriched in intestinal smooth muscle (UniProt) | |
| Isoform / epitope | 2 isoforms; N-terminal processing may affect epitopes (UniProt) |
Section 1
Recommended ACTG2 IHC & IF Protocols
The catalog antibody’s IHC-P protocol is followed by published ACTG2 chromogenic IHC methods for intestinal tumors, soft-tissue sarcoma, and gastric tissue (PMC4841950; PMC11378013; PMC13289488).
| Sample | Paraffin-embedded human colon carcinoma tissue; fixative not specified (datasheet A05066-1) |
| Fixation | Image fixative and duration unreported (datasheet A05066-1); 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 citrate buffer, pH 6.0, 20 min at 95–98 °C (standard rule: cytoplasmic / membrane 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-ACTG2, 1:50-1:200 (datasheet A05066-1) |
| 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 | ACTG2-positive staining in enterocytes - Microvilli of appendix (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression of myoepithelial cells. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6.0 retrieval at 95–98 °C for 20 min (page retrieval). Adjust for the published gastric method if needed (PMC13289488).
Section 2
What Is the Expected ACTG2 Staining Pattern?
ACTG2 is a cytoplasmic cytoskeletal actin with no transmembrane segment (UniProt P63267: location, topology). In intestinal sections, expect staining in smooth muscle cells of the muscularis mucosa, muscularis propria and vessels (UniProt P63267: tissue specificity). HPA reports high staining in smooth muscle cells, but rates its tissue IHC evidence as Supported with medium consistency and cautions that the antibodies can detect proteins from more than one gene (HPA: tissue IHC).
| Strong cytoplasmic staining follows intestinal smooth muscle bundles and vessel walls, with identifiable stained cells and limited background (UniProt P63267: tissue specificity; HPA: High in smooth muscle cells). | This fits the expected ACTG2 distribution in intestinal smooth muscle cells (UniProt P63267: tissue specificity). Judge staining by its compartment and cell type together; intensity alone cannot establish target identity given HPA's multi-gene detection caution (HPA: tissue IHC reliability). |
| Signal is predominantly nuclear or confined to cell surfaces rather than the cytoplasm (UniProt P63267: cytoskeleton and topology). | That compartment disagrees with ACTG2's cytoplasmic cytoskeletal annotation and lack of a transmembrane segment (UniProt P63267: location, topology). Treat it as suspect staining and review morphology and controls before scoring it as ACTG2 (general IHC practice). |
| Strong staining appears in an unexpected cell population, or broad staining obscures the smooth muscle pattern (UniProt P63267: tissue specificity). | Consider antibody cross-reactivity, which is relevant because HPA cautions that its tissue antibodies detect proteins from more than one gene (HPA: tissue IHC reliability). In chromogenic IHC, endogenous detection activity is another possible source; inspect the assay controls before assigning the signal to ACTG2 (general IHC practice). |
| Color is diffuse across tissue, including spaces between cells, without a discernible cytoplasmic smooth muscle pattern (UniProt P63267: location and tissue specificity). | This is background rather than a convincing cellular result (general IHC practice). Compare a no-primary control and tissue morphology; review blocking, washing and chromogen development according to the assay's validated workflow (general IHC practice). |
| Smooth muscle cells show no convincing signal in a known-positive intestinal section (UniProt P63267: tissue specificity; HPA: High in smooth muscle cells). | An absent internal positive pattern makes a negative test result hard to interpret (general IHC practice). Check the positive control, reagent activity and the antibody's validated IHC-P conditions before concluding that the sampled cells lack ACTG2 (general IHC practice). |
💡Expected ACTG2 appearanceCall a positive result when strong cytoplasmic staining maps to identifiable intestinal smooth muscle cells (UniProt P63267: location and tissue specificity; HPA: High in smooth muscle cells); isolated nuclear or indiscriminate tissue-wide color is suspect (UniProt P63267: location; general IHC practice).
| Cell and tissue context (UniProt P63267: tissue specificity; HPA: tissue IHC). | UniProt places abundant ACTG2 in intestinal smooth muscle, including muscularis mucosa, muscularis propria and vascular smooth muscle (UniProt P63267: tissue specificity). HPA also records high smooth muscle staining and high enterocyte-microvillus staining in several intestinal sites (HPA: tissue IHC); interpret these as reported patterns with different levels of target specificity. |
| Antibody attribution (HPA: tissue IHC reliability; HPA: antibodies). | HPA lists two rabbit polyclonal antibodies with IHC status Supported, and describes medium agreement between staining and RNA expression (HPA: HPA041264, HPA041271; tissue IHC reliability). Its explicit multi-gene detection caution limits how confidently any unexpected stained cell can be assigned to ACTG2 alone (HPA: tissue IHC reliability). |
| Compartment and protein forms (UniProt P63267: location, topology, processing, isoforms). | ACTG2 is annotated as cytoplasmic and cytoskeletal, without a transmembrane segment or signal peptide; two isoforms and processed chains are listed (UniProt P63267: location, topology, isoforms, processing). These annotations support a cytoplasmic reading but do not establish antibody epitope coverage or an IHC retrieval requirement. |
| IF/ICC evidence (HPA: subcellular ICC-IF). | Q: What IF/ICC pattern is established here? A: HPA supplies no main subcellular location or cell-line ICC-IF images for ACTG2 (HPA: subcellular ICC-IF). Cytoplasmic localisation is a UniProt annotation, not an image-validated IF pattern in this payload (UniProt P63267: location; HPA: subcellular ICC-IF). |
| Fixation and retrieval evidence (HPA: tissue IHC; UniProt P63267). | Neither supplied source reports a target-specific fixation effect or defines ACTG2 antigen-retrieval conditions (HPA: tissue IHC; UniProt P63267). Use the selected antibody's validated IHC-P instructions to set retrieval conditions, and interpret any adjustment as an assay check rather than an established ACTG2 property (general IHC practice). |
| Situation | Likely cause | Next action |
|---|---|---|
| Expected intestinal smooth muscle is blank (UniProt P63267: tissue specificity; HPA: High in smooth muscle cells). | The run may have failed, or the section may not have yielded detectable signal; these possibilities cannot be distinguished from a blank test slide alone (general IHC practice). | Check an appropriate positive control and the catalog antibody's validated IHC-P instructions, including retrieval and detection steps; repeat only after the control performs as expected (general IHC practice). |
| Nuclei dominate the signal (UniProt P63267: cytoplasmic cytoskeleton). | The observed compartment conflicts with the annotated cytoplasmic location; nonspecific staining or a scoring error is possible (UniProt P63267: location; general IHC practice). | Recheck cell boundaries against the counterstain and compare the no-primary control before scoring the result (general IHC practice). |
| Unexpected cells stain as strongly as smooth muscle (UniProt P63267: tissue specificity). | HPA warns that its antibodies can detect proteins from more than one gene; endogenous chromogenic activity can also produce misleading color (HPA: tissue IHC reliability; general IHC practice). | Compare the stained population with tissue morphology and assay controls; describe unexpected staining as observed signal until its specificity is supported (general IHC practice). |
| The whole section has weak diffuse color that hides cell boundaries (general IHC practice). | Background from the detection workflow may be overwhelming the cellular pattern; the appearance alone does not identify a single failed step (general IHC practice). | Inspect the no-primary control, then review blocking, washes and chromogen development under the validated assay workflow (general IHC practice). |
| Enterocyte microvilli stain strongly in an intestinal section (HPA: High in enterocyte microvilli). | HPA records this pattern in appendix, colon, duodenum, rectum and small intestine, while also cautioning about multi-gene detection (HPA: tissue IHC positive entries and reliability). | Report the microvillus signal separately from smooth muscle staining; do not treat its intensity alone as proof of ACTG2-specific detection (HPA: tissue IHC; general IHC interpretation). |
| A negative cell population stains or an expected negative control becomes positive (HPA: tissue IHC negative entries). | HPA reports no detection in adipocytes and several other listed cell populations, but background or cross-reactivity may alter an assay result (HPA: tissue IHC negative entries and reliability; general IHC practice). | Confirm the cell identity and inspect detection controls; compare with HPA at the cell-population level rather than calling an entire organ ACTG2-negative (HPA: tissue IHC; general IHC practice). |
Experiment evidence
IHC & IF images showing ACTG2 staining
A05066-1 · IHCImmunohistochemistry (IHC) analyzes of Actin-α3/γ2 (E2) pAb in paraffin-embedded human colon carcinoma tissue at 1:50,showing cytoplasmic an
A05066-1 · IHCImmunohistochemistry (IHC) analyzes of Actin-α3/γ2 (E2) pAb in paraffin-embedded human tonsil carcinoma tissue at 1:50,showing cytoplasmic a
A05066 · IHCImmunohistochemistry analysis of paraffin-embedded human skeletal muscle tissue, using Actin-gamma2 Antibody. The picture on the right is bl
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000163017-ACTG2/tissue/cerebral+cortex#imid_12522681
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000163017-ACTG2/tissue/colon#imid_12526415
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000163017-ACTG2/tissue/liver#imid_12526408
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000163017-ACTG2/tissue/kidney#imid_12526425
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for ACTG2 IHC & IF
🧪Run colon first and assess staining in enterocyte microvilli (High; HPA: colon), then run adipose tissue and assess adipocytes (Not detected; HPA: adipose tissue). Do not designate colonic smooth muscle as an internal negative because ACTG2 is reported in intestinal smooth muscle; the supplied HPA row does not establish which other cells on the colon slide should be negative (UniProt P63267 tissue specificity; HPA: colon).
Positive control tissue: Appendix (Enterocytes - Microvilli, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA carries no ICC-IF cell line for ACTG2; 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 a no-primary, secondary-only control and nonimmune IgG matched to the primary antibody’s host and clonality; use ACTG2 knockout material, if available, as a biological specificity control. For chromogenic detection in colon, quench endogenous peroxidase and assess endogenous biotin when using the caption’s avidin–peroxidase detection (selected-SKU caption: goat anti-rabbit IgG-biotin followed by avidin–peroxidase).
⚠️Feasibility: A target-specific fixation window and the effect of antigen retrieval are unreported; the selected-SKU paraffin-section caption does not state the fixative (selected-SKU caption: fixative not stated). The supplied evidence does not establish that frozen sections or IF/ICC are easier; HPA lists no ICC-IF image-bearing cell lines (HPA subcellular). Interpret colon staining cautiously: HPA reports enterocyte microvilli as High, whereas UniProt reports abundant intestinal smooth muscle expression, and the selected-SKU caption also reports nuclear staining despite UniProt’s cytoplasmic cytoskeleton localization (HPA: colon; UniProt P63267 tissue specificity and subcellular location; selected-SKU caption: cytoplasmic and nucleus staining).
HPA tissue IHC evidence for ACTG2
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 → |
| Colon | Enterocytes - Microvilli | High | Protein (IHC) | HPA → |
| Duodenum | Enterocytes - Microvilli | High | Protein (IHC) | HPA → |
| Rectum | Enterocytes - Microvilli | High | Protein (IHC) | HPA → |
| Small intestine | Enterocytes - Microvilli | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Adrenal gland | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | Not detected | Protein (IHC) | HPA → |
| Caudate | Glial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced ACTG2 IHC Tips
Troubleshoot ACTG2 staining in paraffin sections by checking retrieval, cell identity, cytoplasmic localisation and controls before interpreting chromogenic signal.
How should I adjust retrieval when ACTG2 staining is weak or patchy?
Start with citrate buffer at pH 6.0, heated to 95–98 °C for 20 min (page retrieval rule). If staining remains weak, first confirm complete deparaffinisation, consistent section thickness and even heating across slides (standard IHC practice). Then compare a modestly longer retrieval with the page method on adjacent sections, keeping antibody dilution and detection conditions fixed (standard IHC practice). Excess retrieval can damage morphology or increase background, so judge signal alongside preserved tissue structure and a no-primary control (standard IHC practice). Score cytoplasmic staining in intestinal smooth muscle as a reference for the expected pattern (UniProt P63267 tissue specificity; UniProt P63267 subcellular location).
Could fixation explain inconsistent ACTG2 staining between paraffin blocks?
Target-specific fixation sensitivity is unknown because the supplied ACTG2 tissue caption does not state a fixative (A05066-1 tissue-IHC caption). Compare blocks using their recorded fixation histories, section thickness and storage conditions before attributing a signal difference to ACTG2 expression (standard IHC practice). Process a known positive section alongside each batch and hold citrate retrieval at pH 6.0 and 95–98 °C for 20 min constant (page retrieval rule; standard IHC practice). If a block remains weak, optimise retrieval on adjacent sections while checking morphology and no-primary background (standard IHC practice). Do not infer fixation resistance from tissue staining patterns or ACTG2 sequence annotations.
Should ACTG2 staining appear in nuclei or along cell borders?
Expect predominantly cytoplasmic, cytoskeletal staining because ACTG2 is annotated in the cytoplasm and cytoskeleton and lacks a transmembrane segment (UniProt P63267 subcellular location; UniProt P63267 topology). Intestinal muscularis mucosa, muscularis propria and vascular smooth muscle provide anatomically grounded places to assess that pattern (UniProt P63267 tissue specificity). The selected paraffin-section caption reports cytoplasmic and nuclear staining at 1:50, so nuclear signal requires independent scrutiny rather than automatic assignment to ACTG2 (A05066-1 tissue-IHC caption; UniProt P63267 subcellular location). Compare nuclear colour with the no-primary control and check whether it follows tissue folds, edges or damaged cells (standard IHC practice). Report compartments separately when discordant staining persists (standard IHC practice).
How can I assess whether the antibody distinguishes ACTG2 isoforms or related actins?
ACTG2 has 2 annotated isoforms, while actins are highly conserved proteins, so antibody specificity needs direct evidence (UniProt P63267 isoforms; UniProt P63267 function). The supplied caption documents staining in paraffin-embedded human colon carcinoma at 1:50 but does not identify the antibody epitope or establish isoform discrimination (A05066-1 tissue-IHC caption). Request the immunogen sequence or epitope map and compare it with both ACTG2 isoforms and related actins before making an isoform-specific claim (standard antibody-validation practice). Processing at residues 2–3 and annotated modifications mean an N-terminal epitope deserves particular scrutiny if that is where the antibody binds (UniProt P63267 processing; UniProt P63267 modified residues). Include an independent specificity control when conclusions depend on isoform identity (standard IHC practice).
How should I extend this ACTG2 IHC result to multiplex IF?
Treat IF as a separate optimisation: the supplied A05066-1 evidence is a paraffin-section chromogenic IHC image, with no IF fixation or staining conditions reported (A05066-1 tissue-IHC caption). Multiplex ACTG2 with a separately validated smooth muscle cell marker, then check whether cytoplasmic signals overlap in intestinal muscle layers and vascular smooth muscle (UniProt P63267 subcellular location; UniProt P63267 tissue specificity; standard IF practice). Choose fluorophores after measuring tissue autofluorescence, favouring a channel with clearer separation, and include single-stain and no-primary controls (standard IF practice). Because ACTG2 lacks a transmembrane segment and occupies the cytoskeleton, optimise permeabilisation for intracellular access while preserving cell structure (UniProt P63267 topology; UniProt P63267 subcellular location; standard IF practice). Do not transfer the caption's 1:50 IHC dilution to IF without titration (A05066-1 tissue-IHC caption; standard IF practice).
What should I check when DAB obscures ACTG2-positive smooth muscle?
Run a no-primary section through the same detection steps to locate signal caused by secondary reagents, endogenous peroxidase or tissue pigment (standard IHC practice). The selected image used PBS instead of primary antibody as its negative control, followed by biotin-based secondary detection and avidin-peroxidase (A05066-1 tissue-IHC caption). For routine chromogenic IHC, check peroxidase blocking, antibody dilution, wash stringency and DAB development time one variable at a time (standard IHC practice). Compare background in smooth muscle with nearby structures and with the matched negative control; true ACTG2 assessment should prioritise cytoplasmic staining in the expected cells (UniProt P63267 subcellular location; UniProt P63267 tissue specificity; standard IHC practice). Record any diffuse or edge-heavy colour separately from cell-associated signal (standard IHC practice).
How should I score ACTG2 IHC across sections with different muscle content? ⚠ ANSWER MARKED FOR VERIFICATION
Define the scoring compartment before analysis: ACTG2 is cytoplasmic and is abundant in intestinal muscularis mucosa and muscularis propria smooth muscle cells (UniProt P63267 subcellular location; UniProt P63267 tissue specificity). Within annotated smooth muscle regions, report the percentage of positive cells and an intensity-based H-score, using the same threshold and scoring rules across sections (standard IHC practice). Normalise positive-cell counts to the number of evaluable smooth muscle cells, or report stained area per mm² of annotated smooth muscle, rather than per whole-section area (standard IHC practice). Exclude folds, necrosis and damaged edges using prespecified criteria, and review a matched no-primary section for background (standard IHC practice). Keep vascular smooth muscle and intestinal muscle layers as separate regions when their abundance differs (UniProt P63267 tissue specificity; standard IHC practice).
How can I distinguish genuine ACTG2 positivity from staining artefacts?
A plausible ACTG2 pattern is cell-associated cytoplasmic signal in intestinal smooth muscle, including muscularis mucosa, muscularis propria or vascular smooth muscle (UniProt P63267 subcellular location; UniProt P63267 tissue specificity). Treat isolated nuclear colour cautiously: the selected caption reports nuclear staining, but ACTG2's annotated location is cytoplasmic and cytoskeletal (A05066-1 tissue-IHC caption; UniProt P63267 subcellular location). Compare suspicious staining with the no-primary control, especially around tissue edges, folds and necrotic regions, and assess endogenous peroxidase during chromogenic detection (standard IHC practice). Interpret staining outside expected cells conservatively because the HPA tissue-IHC profile cautions that its antibody targets protein from more than one gene (HPA tissue-IHC reliability description). Confirm consequential findings with an independent antibody or orthogonal assay (standard IHC practice).
Boster reagents
Best ACTG2 / Actin, gamma-enteric smooth muscle IHC Antibodies
Two anti-ACTG2 antibodies have paraffin-section IHC images (catalog IHC captions) and list IF use (catalog applications); both list human, mouse, and rat reactivity (catalog reactivity).
Real IHC data
Anti-Actin-Alpha 3/ Gamma 2 (E2) ACTG2 Antibody
Cat # A05066-1
Real IHC data
Anti-Actin alpha3 ACTG2 Antibody
Cat # A05066
A05066-1 will render with a paraffin-embedded human colon carcinoma IHC image at 1:50 and a PBS-for-primary control (A05066-1 IHC image caption). A05066 will render with a paraffin-embedded human skeletal muscle IHC image and a peptide-blocked comparison (A05066 IHC image caption).
Which to pick: For tissue IHC, choose A05066-1 when its colon carcinoma image and PBS control match the planned evaluation; A05066 offers a skeletal muscle image with peptide blocking (respective IHC image captions). For IF/ICC, both list IF, but neither has an IF image in the payload; A05066 lists 1:200–1:1000 and A05066-1 lists 1:50–1:200 (catalog applications and dilutions). Both list human, mouse, and rat reactivity, so neither has a cross-species advantage on the supplied evidence; both IHC captions describe paraffin-embedded sections without reporting the fixative (catalog reactivity; respective IHC image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P63267 (ACTH_HUMAN, Actin, gamma-enteric smooth muscle).
- Human Protein Atlas. ACTG2 tissue IHC expression (reliability: Supported).
- Human Protein Atlas. ACTG2 subcellular location (ICC-IF): Highest expression in U-251MG: 111.8 nTPM.
- Human Protein Atlas. ACTG2 antibody validation summary (2 antibodies).
- A plausible role for actin gamma smooth muscle 2 (ACTG2) in small intestinal neuroendocrine tumorigenesis. BMC endocrine disorders 2016 — PMC4841950.
- Analysis of metastasis‑related risk factors and clinical relevance in adult soft‑tissue sarcoma. Oncology letters 2024 — PMC11378013.
- Actin gamma smooth muscle 2 drives proliferation, metastasis, and 5-Fluorouracil resistance in gastric cancer: insights from a Recurrence-Related Gene Signature. Cancer cell international 2026 — PMC13289488.
- Clinically Relevant Molecular Subtypes in Leiomyosarcoma. Clinical cancer research : an official journal of the American Association for Cancer Research 2015 — PMC4526352.
- PubMed PMID:2377475 — UniProt-cited evidence.
- PubMed PMID:1710027 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for ACTG2 (Actin, gamma-enteric smooth muscle). Expected localisation Cytoplasmic in myoepithelial cells (HPA tissue IHC). Antigen retrieval: Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen). Validated IHC antibody: A05066-1 at 1:50-1:200 (the product's own IHC validation image is shown). Controls (HPA): positive Appendix (Enterocytes - Microvilli); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human colon carcinoma tissue; fixative not specified (datasheet A05066-1); 2. Fixation: Image fixative and duration unreported (datasheet A05066-1); 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 citrate buffer, pH 6.0 , 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-ACTG2, 1:50-1:200 (datasheet A05066-1); 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 A05066-1); Caveat: Staining may reflect proteins from more than one gene (HPA tissue IHC); Isoform / epitope: 2 isoforms; N-terminal processing may affect epitopes (UniProt); Regulation: Enriched in intestinal smooth muscle (UniProt). Evidence: UniProt P63267, HPA tissue IHC · Supported, HPA cell-line RNA · Highest expression in U-251MG: 111.8 nTPM, Datasheet A05066-1, PMC4841950, PMC11378013. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/ACTG2/immunohistochemistry-ihc-guide.