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CFL2 / Cofilin-2 · IHC design guide
Design Immunohistochemistry for CFL2
Plan CFL2 paraffin-section IHC using muscle-rich positive tissue and an IHC-validated antibody (HPA tissue IHC; datasheet M04773). This guide covers expected cytoplasmic staining, controls and interpretation of isoform-related uncertainty (HPA tissue IHC; UniProt).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
CFL2 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; nuclear matrix also annotated (HPA tissue IHC; UniProt) | |
| Staining pattern | Muscle cells show abundant cytoplasmic staining (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6 HIER, heat-mediated (datasheet M04773) | |
| Positive control | Cerebellum+4 more · see all | |
| Negative control | Bone marrow+4 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep paraffin-section fixation consistent across samples. (standard IHC practice; not target-specific) | |
| Caveat | Staining and RNA have medium consistency (HPA tissue IHC) | |
| Regulation | CFL2b predominates in muscle and heart (UniProt) | |
| Isoform / epitope | Three isoforms; isoform-specific epitope coverage is unknown (UniProt) |
Section 1
Recommended CFL2 IHC & IF Protocols
The catalog antibody’s IHC-P protocol is accompanied by published CFL2 IHC methods for colorectal, breast, and sarcoma tissues (PMC11443709; PMC3599769; PMC3440303).
| Sample | Paraffin-embedded human lung cancer tissues; fixative not specified (datasheet M04773) |
| Fixation | Image fixative and duration unreported (datasheet M04773); 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, 20 min (datasheet M04773) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet M04773) |
| Primary antibody | Mouse monoclonal (clone 8C13) anti-CFL2, 0.5-1μg/ml (datasheet M04773) |
| Primary incubation | Overnight at 4 °C (datasheet M04773) |
| Detection | Streptavidin-biotin complex (SABC), DAB chromogen (datasheet M04773) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | CFL2-positive staining in granular cells - cytoplasm/membrane of cerebellum (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression in several tissues, most abundant in muscle tissue. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6 heat retrieval for the catalog antibody (datasheet M04773); the sarcoma study also reports citrate pretreatment (PMC3440303).
Section 2
What Is the Expected CFL2 Staining Pattern?
CFL2 should show predominantly cytoplasmic staining in tissue IHC, strongest in cardiac, skeletal, and smooth muscle cells; cerebellar granular cells may show cytoplasmic and membrane staining (HPA tissue IHC). UniProt also places CFL2 in the cytoskeleton and nuclear matrix, with Z-line colocalization in sarcomeres (UniProt Q9Y281). It has no transmembrane segment (UniProt Q9Y281). HPA rates its tissue IHC reliability Enhanced, while reporting medium agreement with RNA expression (HPA tissue IHC).
| Strong cytoplasmic staining in cardiomyocytes or skeletal myocytes, with stained smooth muscle cells. | This matches the High staining reported for all three cell types; muscle is the most abundant tissue expression in the HPA profile (HPA tissue IHC). Judge the stained cells and compartment together, since a dark section alone does not identify a correct pattern (general IHC practice). |
| Signal confined to an unexpected compartment, with little staining in the expected muscle-cell cytoplasm. | Treat an isolated extracellular or luminal deposit as a possible staining artefact and review the negative control (general IHC practice). Some membrane-associated signal in cerebellar granular cells is reported, however (HPA tissue IHC); compartment calls should therefore be tissue-specific. |
| Strong staining in lung alveolar cells or bone-marrow hematopoietic cells. | HPA reports CFL2 as Not detected in those cell populations (HPA tissue IHC). Review cell identification and controls; widespread signal there may reflect cross-reactivity or endogenous detection activity (general IHC practice). A discrepant stain alone does not establish which cause applies. |
| Diffuse colour across cells and surrounding tissue, obscuring cell boundaries. | This is background until a cell-specific pattern can be resolved (general IHC practice). Compare the primary-antibody-omission control and inspect blocking, washes, and detection chemistry (general IHC practice); HPA's tissue profile cannot assign a cause to background in an individual slide. |
| No detectable signal in heart or skeletal muscle despite an intact section. | These are known High-staining populations in HPA tissue IHC (HPA tissue IHC). Check whether the positive control and detection reagents worked, then review the antibody's IHC-P conditions (general IHC practice). Absence of colour alone does not show that CFL2 is biologically absent. |
💡Expected CFL2 appearanceA convincing positive is High, mainly cytoplasmic staining in cardiac or skeletal myocytes, with smooth muscle cells also High (HPA tissue IHC); diffuse colour without identifiable positive cells is background, while strong staining of HPA Not detected cell populations warrants investigation (general IHC practice; HPA tissue IHC).
| Tissue and cell identity | Cerebellar granular cells are High with cytoplasmic/membrane staining; kidney tubular cells and hepatocytes are Medium (HPA tissue IHC). Compare like cell types when judging intensity (general IHC practice). |
| Isoforms | CFL2b predominates in skeletal muscle and heart, whereas CFL2a occurs in various tissues (UniProt Q9Y281). These expression descriptions do not establish which isoform an unspecified antibody detects. |
| Protein location and processing | CFL2 has no transmembrane segment, signal peptide, or propeptide; the annotated chain spans residues 2–166 (UniProt Q9Y281). These annotations do not establish antibody epitope access in paraffin sections. |
| Tissue IHC confidence | HPA reports Enhanced tissue IHC reliability but medium consistency with RNA data (HPA tissue IHC). Antibody HPA045599 is IHC Enhanced (HPA antibodies); assess a new slide against controls as well as the reference pattern (general IHC practice). |
| Q: What should IF/ICC show? | A: HPA reports mainly plasma-membrane localization, with additional nucleoplasmic and cytosolic signal (HPA ICC-IF). Its ICC-approved antibodies are HPA077782 and CAB037078 (HPA antibodies). This observation comes from ICC-IF and does not replace the tissue IHC pattern. |
| Situation | Likely cause | Next action |
|---|---|---|
| Positive muscle control is blank. | A failed stain or detection step is possible (general IHC practice); HPA reports High signal in cardiac and skeletal myocytes (HPA tissue IHC). | Confirm the control contains the expected cells, then check the IHC-validated antibody, secondary detection, and chromogen against the established IHC-P workflow (general IHC practice). Do not infer CFL2 loss from this slide alone. |
| All tissue compartments appear brown. | Non-specific background or endogenous detection activity can obscure cell-specific staining (general IHC practice). | Compare a primary-antibody-omission control, review blocking and washes, and address endogenous activity appropriate to the detection chemistry (general IHC practice). Reassess only after muscle-cell cytoplasm can be distinguished. |
| A HPA Not detected cell population stains strongly. | Cross-reactivity, endogenous activity, or mistaken cell identification are possible explanations (general IHC practice); HPA lists lung alveolar cells and bone-marrow hematopoietic cells as Not detected (HPA tissue IHC). | Verify the cell type and compare negative and positive controls; investigate persistent discordance with an independently validated IHC antibody if available (general IHC practice). |
| Signal appears only in nuclei of muscle cells. | UniProt includes the nuclear matrix among CFL2 locations, but HPA describes tissue staining chiefly as cytoplasmic (UniProt Q9Y281; HPA tissue IHC). An exclusively nuclear muscle pattern is therefore discordant with the reported tissue profile. | Inspect matched controls and repeat localization assessment with identifiable myocytes (general IHC practice). Record the nuclear finding separately instead of scoring it as the expected cytoplasmic positive. |
| Kidney or liver stains less intensely than muscle. | HPA scores tubular cells and hepatocytes Medium, versus High in cardiac and skeletal myocytes (HPA tissue IHC). | Score the named cell populations against an on-run muscle positive control (general IHC practice). Lower intensity in these tissues alone is consistent with the HPA pattern. |
| ICC-IF membrane signal seems inconsistent with paraffin-section IHC. | The assays have different reported observations: HPA ICC-IF is mainly plasma-membrane localized, while HPA tissue IHC is chiefly cytoplasmic (HPA ICC-IF; HPA tissue IHC). | Interpret each result against its own assay and antibody validation (general IHC practice; HPA antibodies). Use the tissue IHC profile for the paraffin-section decision; consult the separate IF/ICC guide for that assay. |
Experiment evidence
IHC & IF images showing CFL2 staining
M04773 · IHCIHC analysis of Cofilin-2 using anti-Cofilin-2 antibody (M04773). Cofilin-2 was detected in paraffin-embedded section of human lung cancer t
M04773 · IHCIHC analysis of Cofilin-2 using anti-Cofilin-2 antibody (M04773). Cofilin-2 was detected in paraffin-embedded section of human skeletal musc
M04773 · IFIF analysis of Cofilin-2 using anti-Cofilin-2 antibody (M04773). Cofilin-2 was detected in immunocytochemical section of U20S cell. Enzyme a
RP1107 · IFIF analysis of Cofilin 2 using anti-Cofilin 2 antibody (RP1107). Cofilin 2 was detected in an immunocytochemical section of U2OS cells. Enzy
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000165410-CFL2/tissue/cerebral+cortex#imid_17054711
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000165410-CFL2/tissue/colon#imid_17054637
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000165410-CFL2/tissue/liver#imid_17054646
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000165410-CFL2/tissue/kidney#imid_17054631
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody CAB037078; green = CFL2, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody CAB037078; green = CFL2, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for CFL2 IHC & IF
🧪Run skeletal muscle first and require staining in myocytes (HPA: High in skeletal-muscle myocytes). Use bone marrow hematopoietic cells as the negative tissue (HPA: Not detected in bone-marrow hematopoietic cells); on the positive section, assess nonmyocyte areas against background controls rather than assuming those cells are CFL2-negative.
Positive control tissue: Cerebellum (Granular cells - cytoplasm/membrane, HPA High)
Negative control tissue: Bone marrow (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show CFL2 in A-431, U-251MG, U2OS, OE19, with annotated localisation: Plasma membrane (approved) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control; a mouse IgG isotype control matched to the primary antibody’s clonality if known (caption: mouse anti-Cofilin-2); and a CFL2-knockout specimen, if available, as a biological specificity control. For skeletal muscle, quench endogenous peroxidase and check for endogenous biotin before using the reported SABC/DAB detection (caption: SABC/DAB; HPA: High in skeletal-muscle myocytes).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected M04773 paraffin-section caption does not state a fixative (caption: fixative unreported). The caption reports citrate pH 6 heat retrieval for 20 minutes, but does not establish that retrieval is required (caption: citrate pH 6, 20 minutes). The supplied evidence does not establish that frozen sections or IF are easier; endogenous biotin may complicate the reported SABC method in muscle, so assess it with the detection controls (caption: SABC/DAB; HPA: High in skeletal-muscle myocytes).
HPA tissue IHC evidence for CFL2
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — Medium consistency between antibody staining and RNA expression data.). Rows are taken directly from the HPA tissue chart — click any row's HPA link to view the source.
Positive expression · recommended positive controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Cerebellum | Granular cells - cytoplasm/membrane | High | Protein (IHC) | HPA → |
| Heart muscle | Cardiomyocytes | High | Protein (IHC) | HPA → |
| Skeletal muscle | Myocytes | High | Protein (IHC) | HPA → |
| Smooth muscle | Smooth muscle cells | High | Protein (IHC) | HPA → |
| Adrenal gland | Glandular cells | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Bone marrow | Hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Breast | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | Not detected | Protein (IHC) | HPA → |
| Esophagus | Squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
| Hippocampus | Glial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced CFL2 IHC Tips
Troubleshoot CFL2 staining in paraffin-section chromogenic IHC using the catalog antibody’s tissue example, with IF/ICC considerations kept separate.
Which retrieval conditions should I try first for weak CFL2 staining?
Start with heat-mediated citrate retrieval at pH 6 for 20 minutes (datasheet M04773). This is the condition reported for the catalog antibody in a paraffin-embedded human lung cancer section, although the caption does not state the fixative (datasheet M04773). If staining remains weak, compare retrieval time or a different buffer on matched sections while holding antibody concentration and detection constant; evaluate morphology alongside signal (standard IHC practice). Include a known positive muscle section to distinguish retrieval failure from genuinely sparse expression in the test tissue (HPA: high in skeletal muscle myocytes). Record both signal and nonspecific staining before adopting a stronger condition (standard IHC practice).
Could fixation explain inconsistent CFL2 staining between paraffin blocks?
Target-specific CFL2 sensitivity to fixation is unknown from the supplied evidence: the catalog tissue caption identifies a paraffin-embedded section but does not report its fixative (datasheet M04773). Record each block’s fixative, fixation duration, and processing history before comparing staining intensity (standard IHC practice). Test suspect blocks beside a consistently positive control using the same citrate pH 6, 20-minute retrieval and detection run (datasheet M04773; standard IHC practice). Differences that track block preparation warrant a controlled fixation study; staining patterns alone cannot establish which preparation step altered the epitope (standard IHC practice). Keep tissue morphology in the review so damaged sections are not scored as true loss of CFL2 (standard IHC practice).
Should CFL2 staining appear in cytoplasm, at membranes, or in nuclei?
Expect substantial cytoplasmic staining in tissue IHC, especially in muscle, while assessing the specific cell type being scored (HPA: cytoplasmic expression in several tissues, most abundant in muscle). CFL2 is associated with the cytoskeleton and nuclear matrix, and its record places it with CSRP3 at sarcomeric Z lines (UniProt Q9Y281 localization). Separate ICC/IF evidence reports plasma membrane as the main location, with nucleoplasm and cytosol also approved (HPA subcellular). Treat a thin membrane rim or focal nuclear signal as a pattern to verify with controls and appropriate imaging, rather than automatically rejecting it (HPA subcellular; standard IHC practice). Diffuse staining across every compartment or cell type calls for background assessment (standard IHC practice).
Can this stain distinguish CFL2 isoforms or phosphorylation states?
Do not assign isoform or phosphorylation state from this stain unless the antibody’s epitope and cross-reactivity have been established independently (standard IHC practice). CFL2 has 3 reported isoforms; CFL2b predominates in skeletal muscle and heart, whereas CFL2a occurs in various tissues (UniProt Q9Y281 isoforms and tissue specificity). Its ADF-H domain spans residues 4–153, and reported modifications include phosphoserine at residue 3 and phosphothreonine at residue 6 (UniProt Q9Y281 domains and modified residues). These facts make epitope mapping relevant, but they do not establish what M04773 recognizes (UniProt Q9Y281; datasheet M04773). Compare validated isoform-specific or modification-specific reagents before making those claims (standard IHC practice).
How should I assess CFL2 by multiplex IF after developing the IHC stain?
Develop IF/ICC as a separate assay: the supplied M04773 tissue example documents chromogenic IHC on a paraffin section, with no IF fixation or staining conditions (datasheet M04773). Pair CFL2 with a marker identifying the expected cell type, such as a validated cardiomyocyte marker in heart, and include single-channel controls (HPA: high in cardiomyocytes; standard IF practice). Choose spectrally separated fluorophores and consider a far-red CFL2 channel when tissue autofluorescence obscures shorter wavelengths (standard IF practice). Because CFL2 has no transmembrane segment and has cytosolic and nuclear locations, optimize permeabilisation for access to intracellular epitopes while preserving morphology (UniProt Q9Y281 topology and localization; standard IF practice). Confirm apparent overlap against bleed-through controls before calling colocalisation (standard IF practice).
What should I check when DAB staining spreads across the section?
Compare the stained section with a primary-omission control and inspect whether color follows tissue edges, damaged areas, or all cell types indiscriminately (standard IHC practice). The catalog example used 10% goat serum blocking, 1 μg/ml primary antibody overnight at 4°C, and biotin-based DAB detection (datasheet M04773). Check peroxidase blocking, secondary-only reactivity, and any background associated with the biotin detection system as general chromogenic IHC controls (standard IHC practice). Titrate the primary and shorten development if background rises faster than plausible cell-associated signal (standard IHC practice). Compare with a high-expression muscle control and a tissue compartment reported as undetected, such as lung alveolar cells (HPA tissue IHC).
How can I quantify CFL2 staining without confounding tissue composition? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring, then keep the same thresholds and imaging settings across matched sections (standard IHC practice). For cellular DAB staining, report the percentage of positive target cells and an H-score based on staining intensity; use positive-cell density per mm² when cell abundance itself is the endpoint (standard IHC practice). Normalize counts to the area or number of eligible cells, excluding empty space, necrosis, and damaged edges (standard IHC practice). Separate myocytes from other cells in muscle, because CFL2 is abundant there and cell composition can dominate section-wide averages (HPA: high in skeletal muscle myocytes; standard IHC practice). Include control-section performance and the scoring rules with each comparison (standard IHC practice).
How do I distinguish genuine CFL2 positivity from staining artefact?
Look for reproducible cell-associated staining in an expected population, such as skeletal myocytes or cardiomyocytes, with a plausible cytoplasmic distribution (HPA tissue IHC). Interpret a lung cancer section cautiously: the catalog shows detection there, while normal lung alveolar cells were reported as undetected in the HPA tissue panel (datasheet M04773; HPA tissue IHC). A signal confined to section edges, necrosis, or many unrelated compartments suggests processing or detection artefact and needs control review (standard IHC practice). Check primary-omission and peroxidase controls before accepting widespread DAB color as CFL2 (standard IHC practice). If nuclear or membrane staining drives the conclusion, corroborate its cell specificity because those locations have separate support from UniProt and ICC/IF evidence (UniProt Q9Y281 localization; HPA subcellular).
Boster reagents
Best CFL2 / Cofilin-2 IHC Antibodies
Real CFL2 IHC images cover paraffin sections of human lung cancer and human, mouse, and rat skeletal muscle (M04773 IHC captions); an IF image covers U2OS cells (RP1107 IF caption).
Real IHC data
Anti-Cofilin 2/CFL2 Antibody ® (monoclonal, 8C13)
Cat # M04773
Real IF data
Anti-Cofilin 2/CFL2 Antibody ®
Cat # RP1107
M04773 is listed for IHC and IF (catalog applications), with paraffin-section images from human lung cancer and human, mouse, and rat skeletal muscle (M04773 IHC captions). RP1107 is listed for IF/ICC (catalog applications), with an IF image in U2OS cells (RP1107 IF caption).
Which to pick: Choose M04773 for paraffin-section tissue IHC: its monoclonal antibody is listed for IHC (M04773 catalog), and its IHC captions show 1 μg/ml staining after citrate retrieval (M04773 IHC captions); the fixative is unreported (M04773 IHC captions). Choose RP1107 for IF/ICC when its U2OS cell image fits the experiment (RP1107 catalog applications; RP1107 IF caption). For tissue IHC across human, mouse, and rat, M04773 has images in all three species (M04773 IHC captions); RP1107 lists all three as reactive, but its supplied IF image shows U2OS cells (RP1107 catalog reactivity; RP1107 IF caption).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q9Y281 (COF2_HUMAN, Cofilin-2).
- Human Protein Atlas. CFL2 tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. CFL2 subcellular location (ICC-IF): Mainly localized to the plasma membrane. In addition localized to the nucleoplasm and cytosol..
- Human Protein Atlas. CFL2 antibody validation summary (3 antibodies).
- USP14 promotes head and neck squamous cell carcinoma progression via deubiquitinating and stabilizing CFL2. Future science OA 2026 — PMC13374754.
- Comprehensive evaluation of immunological attributes and immunotherapy responses of positive T cell function regulators in colorectal cancer. BMC gastroenterology 2024 — PMC11443709.
- A systematic evaluation of miRNA:mRNA interactions involved in the migration and invasion of breast cancer cells. Journal of translational medicine 2013 — PMC3599769.
- Expression of subtype-specific group 1 leiomyosarcoma markers in a wide variety of sarcomas by gene expression analysis and immunohistochemistry. The American journal of surgical pathology 2011 — PMC3440303.
- PubMed PMID:11422377 — UniProt-cited evidence.
- PubMed PMID:12508121 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for CFL2 (Cofilin-2). Expected localisation Cytoplasmic tissue staining; nuclear matrix also annotated (HPA tissue IHC; UniProt). Antigen retrieval: Citrate pH 6 HIER, heat-mediated (datasheet M04773). Validated IHC antibody: M04773 at 0.5-1μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Cerebellum (Granular cells - cytoplasm/membrane); negative Bone marrow. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human lung cancer tissues; fixative not specified (datasheet M04773); 2. Fixation: Image fixative and duration unreported (datasheet M04773); 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 , 20 min (datasheet M04773); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet M04773); 8. Primary antibody: Mouse monoclonal (clone 8C13) anti-CFL2, 0.5-1μg/ml (datasheet M04773); 9. Primary incubation: Overnight at 4 °C (datasheet M04773). Watch for: Fixation: Keep paraffin-section fixation consistent across samples. (standard IHC practice; not target-specific); Caveat: Staining and RNA have medium consistency (HPA tissue IHC); Isoform / epitope: Three isoforms; isoform-specific epitope coverage is unknown (UniProt); Regulation: CFL2b predominates in muscle and heart (UniProt). Evidence: UniProt Q9Y281, HPA tissue IHC · Enhanced, HPA ICC-IF · Mainly localized to the plasma membrane. In addition localized to…, Datasheet M04773, PMC11443709, PMC3599769. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/CFL2/immunohistochemistry-ihc-guide.