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CFL2 / Cofilin-2 · IHC design guide
Design Immunohistochemistry for CFL2
Plan CFL2 staining in paraffin sections using skeletal or heart muscle as a high-expression reference (HPA tissue IHC). This guide covers fixation, antibody dilution, chromogenic detection and interpretation of the expected cytoplasmic pattern (HPA tissue IHC).
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 | Predominantly cytoplasmic in tissue (HPA tissue IHC) | |
| Staining pattern | Cytoplasmic staining, most abundant in muscle (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 fixation consistent across sections. (standard IHC practice; not target-specific) | |
| Caveat | Staining has medium concordance with RNA expression (HPA tissue IHC) | |
| Regulation | CFL2b predominates in heart and skeletal muscle (UniProt) | |
| Isoform / epitope | 3 isoforms; check epitope coverage (UniProt) |
Section 1
Recommended CFL2 IHC & IF Protocols
Start with the catalog antibody’s IHC-P protocol (datasheet: M04773), then compare published CFL2 staining in colorectal and breast tissues (PMC11443709; PMC3599769).
| 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 noteTry citrate pH 6 heat retrieval first (datasheet: M04773); the cited articles do not specify retrieval conditions (PMC11443709; PMC3599769).
Section 2
What Is the Expected CFL2 Staining Pattern?
In paraffin-section IHC, expect CFL2 staining chiefly in the cytoplasm of myocytes and cardiomyocytes; HPA also reports high staining in smooth muscle cells and cerebellar granular cells (HPA tissue IHC: Enhanced; medium consistency with RNA). UniProt places CFL2 in the cytoskeleton and nuclear matrix and reports no transmembrane segment (UniProt Q9Y281). HPA's plasma-membrane, nucleoplasm and cytosol calls come from ICC-IF and should be interpreted in that context (HPA subcellular ICC-IF).
| Strong intracellular staining in heart or skeletal muscle cells, with cytoplasmic detail that follows the cells. | This fits the expected IHC result: HPA reports high staining in cardiomyocytes and skeletal myocytes, and UniProt places CFL2 in the cytoskeleton (HPA tissue IHC; UniProt Q9Y281). Assess which cells stain as well as intensity; a brown field alone cannot establish that the signal comes from muscle cells (general IHC interpretation). |
| Staining is confined to an extracellular rim or tissue spaces, without convincing intracellular staining in muscle cells. | Treat this as a suspect compartment pattern, since UniProt describes cytoskeletal and nuclear-matrix localization and no transmembrane segment (UniProt Q9Y281). HPA's approved plasma-membrane call is from ICC-IF, so it does not by itself validate an extracellular-only chromogenic IHC pattern (HPA subcellular ICC-IF). |
| The strongest signal falls in a cell population reported as undetected, while adjacent expected-positive cells are faint. | Check the cell identity before calling cross-reactivity: HPA reports undetected staining in bone-marrow hematopoietic cells and lung alveolar cells, but its calls are cell-specific rather than declarations that every cell in those tissues is negative (HPA tissue IHC). Endogenous detection activity is another possible cause of a chromogenic signal (general IHC practice). |
| Brown deposit spreads across cells and tissue spaces, obscuring boundaries and preventing cell-level scoring. | Score this as uninterpretable background rather than CFL2 positivity, because the expected IHC finding is cell-associated cytoplasmic staining, most abundant in muscle tissue (HPA tissue IHC). Background can arise from nonspecific reagent binding, incomplete blocking or detection chemistry (general IHC practice); its appearance alone does not identify the cause. |
| No detectable staining appears in an otherwise evaluable heart or skeletal-muscle section. | Investigate the run before concluding that CFL2 is absent: HPA reports high staining in cardiomyocytes and skeletal myocytes (HPA tissue IHC). Inspect section integrity and controls, then check the antibody's IHC-P instructions and detection steps (general IHC practice). HPA does not establish a CFL2-specific fixation or retrieval sensitivity. |
💡Expected CFL2 appearanceA convincing positive is strong, cell-associated cytoplasmic staining in heart or skeletal myocytes (HPA tissue IHC: High), whereas extracellular-only deposit or staining dominated by an HPA-undetected cell population warrants investigation (UniProt Q9Y281; HPA tissue IHC).
| Tissue and cell selection | Heart cardiomyocytes and skeletal myocytes are high-staining reference cells; smooth muscle cells and cerebellar granular cells are also reported high (HPA tissue IHC). Kidney tubular cells and hepatocytes are reported medium, so weaker staining there can still match the atlas pattern (HPA tissue IHC). Compare named cell populations, not whole organs. |
| Isoform distribution | UniProt reports CFL2b predominantly in skeletal muscle and heart and CFL2a in various tissues (UniProt Q9Y281). This supports tissue-dependent interpretation, but the supplied IHC record does not establish which isoform an antibody detects in a given section; do not assign isoform identity from staining intensity alone. |
| Compartment and membrane interpretation | UniProt places CFL2 in cytoskeleton and nuclear matrix and reports no transmembrane segment (UniProt Q9Y281). HPA tissue IHC describes cytoplasmic expression, while HPA ICC-IF calls plasma membrane the main location with nucleoplasm and cytosol additional locations (HPA tissue IHC; HPA subcellular ICC-IF). Keep the assay-specific observations distinct. |
| Evidence strength | HPA labels tissue IHC reliability Enhanced, with medium consistency between antibody staining and RNA expression data (HPA tissue IHC). Its antibody table lists Enhanced IHC for HPA045599, while HPA077782 and CAB037078 have Approved ICC calls and no listed IHC status (HPA antibodies). Apply those validation labels to their stated assays. |
| Antigen retrieval and fixation | The supplied sources provide no CFL2-specific fixation effect, retrieval condition or epitope location. Use the IHC-validated antibody's documented paraffin-section instructions and assess run controls when optimizing retrieval (general IHC practice). Do not infer a retrieval requirement from UniProt topology, modified residues or HPA tissue intensity (UniProt Q9Y281; HPA tissue IHC). |
| Situation | Likely cause | Next action |
|---|---|---|
| Heart or skeletal muscle shows no cell-associated signal. | A failed stain or detection step is possible; HPA reports these muscle cells as high-staining, but gives no CFL2-specific fixation explanation (HPA tissue IHC). | Check the section, positive-run control and reagent sequence; then follow the catalog antibody's IHC-P instructions for dilution, retrieval and detection (general IHC practice). Repeat with a controlled change if the run failed. |
| Every tissue compartment has a diffuse brown haze. | Nonspecific binding or excessive detection background may obscure the cell pattern (general IHC practice). HPA's expected tissue profile is cytoplasmic and most abundant in muscle (HPA tissue IHC). | Review blocking, washing and detection development; inspect a no-primary control for reagent-derived background (general IHC practice). Reassess cell-associated staining only after the haze is reduced. |
| An HPA-undetected cell type appears strongly positive. | Cross-reactivity or endogenous chromogenic activity is possible, but cell misidentification can also explain an apparent conflict (general IHC practice; HPA tissue IHC). | Identify the stained cells against morphology, then compare a no-primary control and a separate well-characterized positive tissue (general IHC practice). HPA calls bone-marrow hematopoietic cells undetected; it does not label every bone-marrow structure negative (HPA tissue IHC). |
| Signal is exclusively extracellular or cannot be assigned to intact cells. | That location conflicts with UniProt's intracellular cytoskeleton and nuclear-matrix annotation (UniProt Q9Y281); tissue damage or chromogen deposit can mimic staining (general IHC practice). | Inspect morphology and counterstain, check the no-primary control, and repeat an affected section if needed (general IHC practice). Do not use the ICC-IF plasma-membrane call to justify extracellular-only IHC staining (HPA subcellular ICC-IF). |
| A medium-staining tissue looks weaker than muscle. | The contrast can be expected: HPA reports medium staining in kidney tubular cells and hepatocytes versus high staining in heart and skeletal myocytes (HPA tissue IHC). | Score the relevant cell types within each section and compare them with their own expected HPA levels (HPA tissue IHC). Check the run control before increasing detection intensity, which can raise background (general IHC practice). |
| An IF/ICC image seems to show membrane or nuclear signal that differs from the IHC slide. | HPA ICC-IF approves plasma membrane as the main location and nucleoplasm and cytosol as additional locations; its tissue-IHC summary describes cytoplasmic staining (HPA subcellular ICC-IF; HPA tissue IHC). | Interpret each result against its assay-specific HPA record and cell context (HPA subcellular ICC-IF; HPA tissue IHC). For IF/ICC workflow details, use the separate IF/ICC guide; this section's primary readout is chromogenic paraffin-section IHC. |
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 look for staining in myocytes (HPA: High in skeletal-muscle myocytes). Use bone marrow hematopoietic cells as the negative tissue (HPA: Not detected); on the muscle slide, compare myocytes with nearby non-myocyte areas as a background reference, without treating those areas as a validated negative (HPA: skeletal-muscle myocytes are High).
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 no-primary (secondary-only), a concentration-matched mouse IgG isotype control, and CFL2 knockout material processed in parallel (caption: mouse primary antibody; standard IHC practice). Quench endogenous peroxidase and check for endogenous biotin background if using the caption’s streptavidin–biotin/DAB detection on muscle sections (caption: SABC/DAB; standard IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected M04773 paraffin-section caption does not state the fixative (caption: fixative not stated). The caption uses heat retrieval in citrate buffer at pH 6 for 20 minutes, but does not establish that retrieval is required (caption: citrate retrieval). Neither frozen sections nor IF is established as easier by the supplied evidence; for skeletal muscle, assess any staining from endogenous peroxidase or biotin before scoring myocytes (HPA: High in skeletal-muscle myocytes; caption: SABC/DAB; standard IHC practice).
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 sections by checking retrieval, cell identity, compartment, and controls before interpreting chromogenic signal.
How should I adjust retrieval when CFL2 staining is weak in paraffin sections?
Start with heat-mediated citrate retrieval at pH 6 for paraffin-section IHC (datasheet M04773). The catalog antibody's tissue image used 20 minutes of citrate heating before primary incubation and DAB development (caption M04773). Run a retrieval time series alongside a known positive section, such as skeletal muscle, while keeping detection conditions constant (standard IHC practice; HPA: High in skeletal-muscle myocytes). If signal remains weak, compare a second retrieval condition as a fallback and check whether tissue architecture or background deteriorates (standard IHC practice). Record the condition used for each section before comparing staining intensity (standard IHC practice).
Could fixation explain weak or uneven CFL2 staining?
The selected paraffin-section caption does not state a fixative, so target-specific fixation sensitivity is unknown (caption M04773). Record the actual fixative, fixation duration, and processing history for each block before comparing CFL2 intensity across specimens (standard IHC practice). If fixation histories differ, stain a matched positive control in the same run and interpret differences cautiously (standard IHC practice; HPA: High in heart-muscle cardiomyocytes). Examine poorly preserved areas separately from well-preserved tissue, since damaged morphology complicates cell-level scoring (standard IHC practice). Do not assign an optimal fixation time to CFL2 from the available evidence (caption M04773).
Which cellular compartments should contain credible CFL2 staining?
Expect predominantly cytoplasmic tissue staining, with particularly strong expression reported in muscle tissue (HPA tissue IHC: cytoplasmic profile, most abundant in muscle). UniProt also places CFL2 in the cytoskeleton and nuclear matrix, with Z-line colocalization noted in sarcomeres (UniProt Q9Y281: subcellular location). HPA cell imaging reports an approved plasma-membrane location plus nucleoplasm and cytosol, so compartment calls require cell context (HPA subcellular: approved locations). Compare suspicious staining with morphology, a matched positive section, and a primary-omission control before assigning it to CFL2 (standard IHC practice; HPA: High in skeletal-muscle myocytes). Score compartments separately when patterns differ within a specimen (standard IHC practice).
How can isoforms or epitope masking affect CFL2 interpretation?
CFL2 has 3 listed isoforms, and the supplied record does not map this antibody's epitope or establish isoform selectivity (UniProt Q9Y281: isoforms; caption M04773). CFL2b predominates in skeletal muscle and heart, whereas CFL2a occurs in various tissues, so tissue staining alone cannot identify an isoform (UniProt Q9Y281: tissue specificity). The ADF-H domain spans residues 4–153, and listed N-terminal modifications include phosphorylation and acetylation; their effect on this antibody is unreported (UniProt Q9Y281: domain and modified residues; caption M04773). Compare matched controls under identical retrieval conditions before attributing variable staining to epitope accessibility (standard IHC practice). Request epitope-mapping or isoform-specific validation if that distinction drives the experiment (standard IHC practice).
How should I investigate CFL2 localisation with IF after IHC?
Treat IF as a separate localisation experiment, because the selected antibody evidence here documents paraffin-section chromogenic IHC (caption M04773). Multiplex CFL2 with a marker for the expected cell type, such as a cardiomyocyte marker in heart tissue, and include single-stain controls (HPA: High in cardiomyocytes; standard IF practice). Choose well-separated fluorophores and a detection channel with low tissue autofluorescence, then inspect unstained tissue in each channel (standard IF practice). For a proposed intracellular epitope, test gentle permeabilisation; for a proposed surface-accessible epitope, compare staining without detergent, since this antibody's epitope side is unreported (UniProt Q9Y281: no transmembrane segment; HPA subcellular: plasma membrane, cytosol, nucleoplasm; standard IF practice).
What should I check when DAB background obscures CFL2 staining?
The catalog image used 10% goat serum blocking, a 30-minute biotinylated secondary incubation at 37°C, and DAB detection (caption M04773). Check primary-omission and secondary-only sections to distinguish antibody-dependent signal from detection background (standard IHC practice). For a peroxidase-based DAB workflow, verify the endogenous peroxidase block and inspect whether biotin-based detection contributes diffuse staining (standard IHC practice; caption M04773: biotinylated secondary and SABC). If background persists, titrate primary antibody and detection exposure while processing a positive section in parallel (standard IHC practice). Avoid treating diffuse edge staining or precipitate as intracellular CFL2 without agreement from morphology and controls (standard IHC practice; UniProt Q9Y281: cytoskeleton and nuclear matrix).
How should I quantify CFL2 staining across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring, because CFL2 tissue staining is mainly cytoplasmic while other locations are also reported (HPA tissue IHC: cytoplasmic profile; UniProt Q9Y281: nuclear matrix). For chromogenic sections, record percent positive cells and intensity, then calculate an H-score when intensity categories are reproducible (standard IHC practice). Normalize positive-cell counts or stained area to the number of eligible cells or analyzed tissue area in mm², respectively (standard IHC practice). Apply the same retrieval, imaging, threshold, and exclusion rules across specimens, with a positive control on each staining run (standard IHC practice). Report cell-type strata separately when muscle and nonmuscle cells share a section (HPA: muscle-enriched expression; standard IHC practice).
How can I distinguish genuine CFL2 staining from artefact?
Give greater weight to staining in morphologically intact expected cells, such as skeletal-muscle myocytes or cardiomyocytes, than to isolated signal at section edges (HPA: High in both cell populations; standard IHC practice). Cytoplasmic staining fits the tissue IHC profile, while nuclear or membrane-associated signal needs compartment-aware review against the reported localisation evidence (HPA tissue IHC: cytoplasmic profile; HPA subcellular: nucleoplasm and plasma membrane). Exclude necrotic regions and edge artefacts from scoring, and compare a primary-omission control to assess nonspecific DAB deposition (standard IHC practice). Check endogenous enzyme activity in the peroxidase workflow before calling brown signal antigen-specific (standard IHC practice). Consider HPA's staining-to-RNA consistency only medium when weighing an unexpected cell pattern (HPA tissue IHC: Enhanced reliability, medium consistency).
Boster reagents
Best CFL2 / Cofilin-2 IHC Antibodies
Anti-CFL2 IHC images cover paraffin sections of human lung cancer and human, mouse, and rat skeletal muscle (M04773 IHC captions); IF images cover U2OS cells (M04773 and RP1107 IF captions).
Real IHC data
Anti-Cofilin 2/CFL2 Antibody ® (monoclonal, 8C13)
Cat # M04773
Real IF data
Anti-Cofilin 2/CFL2 Antibody ®
Cat # RP1107
M04773 has IHC images from human lung cancer and human, mouse, and rat skeletal muscle paraffin sections, plus an IF image from U2OS cells (M04773 image captions). RP1107 has an IF/ICC image from U2OS cells and lists human, mouse, and rat reactivity, but does not list IHC as an application (RP1107 IF caption; catalog: applications and reactivity).
Which to pick: Choose M04773 for chromogenic IHC of paraffin sections: it is a mouse monoclonal antibody with IHC images using citrate pH 6 retrieval and DAB detection; the fixative is unreported (catalog: M04773 clone and applications; M04773 IHC captions). Choose RP1107 for IF/ICC when a rabbit primary is preferred; its U2OS IF image supports that application at 5 μg/ml (catalog: RP1107 host and applications; RP1107 IF caption). For cross-species tissue IHC, M04773 has image evidence in human, mouse, and rat sections, whereas RP1107 has no IHC image or listed IHC application (M04773 IHC captions; catalog: RP1107 applications).
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 Predominantly cytoplasmic in tissue (HPA tissue IHC). 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 fixation consistent across sections. (standard IHC practice; not target-specific); Caveat: Staining has medium concordance with RNA expression (HPA tissue IHC); Isoform / epitope: 3 isoforms; check epitope coverage (UniProt); Regulation: CFL2b predominates in heart and skeletal muscle (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.