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FEN1 / Flap endonuclease 1 · IHC design guide
Design Immunohistochemistry for FEN1
Plan FEN1 staining in paraffin sections around the nuclear signal seen in highly proliferative cells (HPA tissue IHC). Compare with a low-signal tissue control and assess nuclear staining across cell populations (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
FEN1 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 | Nuclear staining in tissue; nucleolar enrichment is a molecular expectation (HPA tissue IHC; UniProt) | |
| Staining pattern | Nuclear signal in highly proliferative cells (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet M01484-3) | |
| Positive control | Adrenal gland+4 more · see all | |
| Negative control | Heart muscle+3 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 | Cardiomyocytes may be unstained (HPA tissue IHC) | |
| Regulation | Enriched in highly proliferative cells (HPA tissue IHC) | |
| Isoform / epitope | 2 isoforms, FEN1 and FENMIT; epitope coverage unknown (UniProt) |
Section 1
Recommended FEN1 IHC & IF Protocols
The catalog antibody protocol uses heat retrieval in EDTA pH 8.0 (datasheet M01484-3). The published IHC protocols below cover osteosarcoma and hepatocellular carcinoma (PMC8504935; PMC8771828; PMC7201445).
| Sample | Paraffin-embedded human bladder epithelial carcinoma tissue; fixative not specified (datasheet M01484-3) |
| Fixation | Image fixative and duration unreported (datasheet M01484-3); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: EDTA pH 8.0 (datasheet M01484-3); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet M01484-3) |
| Primary antibody | Mouse monoclonal (clone 6F3F2) anti-FEN1, 2 μg/ml (datasheet M01484-3) |
| Primary incubation | Overnight at 4 °C (datasheet M01484-3) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet M01484-3) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | FEN1-positive staining in glandular cells of adrenal gland (HPA tissue IHC: High). HPA tissue profile: Nuclear expression in highly proliferative cells in most tissues. No signal in the no-primary control. |
💡Decision noteStart with EDTA pH 8.0 for the catalog antibody (datasheet M01484-3). Try citrate pH 6.0 when adapting the osteosarcoma protocol (PMC8504935).
Section 2
What Is the Expected FEN1 Staining Pattern?
FEN1 should stain nuclei, especially in highly proliferative cells across most tissues (HPA tissue IHC: Enhanced). UniProt places FEN1 in the nucleolus and nucleoplasm, also annotates mitochondria, and reports no transmembrane segment (UniProt P39748). Interpret chromogenic staining by both compartment and cell type: HPA reports high staining in lymph node germinal center cells and bone marrow hematopoietic cells, while cardiomyocytes are not detected (HPA tissue IHC).
| Distinct nuclear staining in germinal center cells, with weaker or absent staining in surrounding cells (HPA tissue IHC). | This fits the reported nuclear pattern in highly proliferative cells and high staining in lymph node germinal centers (HPA tissue IHC). Judge the stained cell population, not the whole section: variation between neighboring cells can be expected from the HPA profile, so a uniformly positive field is not required. |
| Predominantly cytoplasmic or membranous color, with little convincing nuclear staining. | That distribution conflicts with the principal nuclear pattern in tissue IHC (HPA tissue IHC) and with the absence of a transmembrane segment (UniProt P39748). UniProt also annotates mitochondria, so extranuclear signal alone cannot prove an artefact; compare a known nuclear-positive tissue and reassess detection background before assigning it to FEN1. |
| Strong staining in cardiomyocytes or skeletal myocytes, or uniformly strong staining across unrelated cell types. | HPA reports FEN1 as not detected in cardiomyocytes and skeletal myocytes, while its overall tissue profile emphasizes highly proliferative cells (HPA tissue IHC). Confirm which cells carry the color; unexpected staining warrants checks for antibody cross-reactivity and endogenous detection activity (general IHC practice). |
| Diffuse color covers nuclei, cytoplasm, and surrounding tissue without clear cell boundaries. | This cannot establish the nuclear FEN1 pattern reported by HPA (HPA tissue IHC). Review a negative detection control, blocking, washing, and chromogen development to locate nonspecific or endogenous signal (general IHC practice); do not score diffuse background as FEN1-positive cells. |
| No nuclear signal in germinal center cells or bone marrow hematopoietic cells. | Both populations have high FEN1 staining in HPA tissue IHC (HPA tissue IHC). An entirely blank result in a suitable positive section calls for a workflow check, including antibody preparation, antigen retrieval, and detection controls (general IHC practice), before interpreting the sample as FEN1-negative. |
💡Expected FEN1 appearanceCall a result positive when chromogenic signal resolves within nuclei of the expected cell population, prominently in highly proliferative cells (HPA tissue IHC: nuclear profile), with high staining possible in lymph node germinal center cells (HPA tissue IHC: High); diffuse pan-tissue color or dominant membrane staining should prompt a background or specificity check (general IHC practice; UniProt P39748 topology).
| Nuclear compartment | HPA tissue IHC describes nuclear expression, while UniProt places FEN1 in nucleoli and nucleoplasm and notes movement toward the nucleoplasm after DNA damage (HPA tissue IHC; UniProt P39748). Do not require every positive nucleus to display a separately resolved nucleolus in a chromogenic section (general IHC practice). |
| Cell population and tissue choice | High staining in colon glandular cells and lymph node germinal center cells contrasts with FEN1 not being detected in cardiomyocytes (HPA tissue IHC). Choose and score the named cell population rather than using all cells in either tissue as a single positive or negative control. |
| Antibody evidence | HPA marks tissue IHC reliability as Enhanced and lists Enhanced IHC validation for HPA006748 and CAB002262 (HPA tissue IHC; HPA antibodies). That supports the reported pattern; it does not establish the staining behavior of an unspecified catalog antibody under the reader’s staining conditions. |
| Isoforms and mitochondrial annotation | UniProt lists two isoforms and a mitochondrial location alongside nuclear locations (UniProt P39748). The supplied record gives no antibody epitope or isoform-specific IHC pattern, so it cannot determine which isoform an unexpected extranuclear signal represents or validate that signal by location alone. |
| IF/ICC Q&A: where should signal appear? | Mainly in the nucleoplasm, with additional nucleolar signal (HPA subcellular ICC-IF: supported). This is an IF/ICC localization answer; tissue IHC interpretation still follows the nuclear pattern and cell populations reported for sections (HPA tissue IHC). |
| Fixation sensitivity | Target-specific fixation sensitivity is unreported in the supplied UniProt and HPA records. Record the fixation and retrieval conditions used for comparisons (general IHC practice), without attributing a change in FEN1 staining to fixation from these sources. |
| Situation | Likely cause | Next action |
|---|---|---|
| A putative positive section shows no nuclear color. | The expected cell population may be absent, or an antibody, retrieval, or detection step may have failed (HPA tissue IHC; general IHC practice). | Locate germinal center cells or bone marrow hematopoietic cells, both reported High (HPA tissue IHC); then check reagent preparation, retrieval, and detection with suitable controls (general IHC practice). |
| Color is mostly cytoplasmic or along cell borders. | The dominant pattern is inconsistent with HPA nuclear tissue staining; FEN1 has no transmembrane segment, although UniProt also annotates mitochondria (HPA tissue IHC; UniProt P39748). | Compare a known nuclear-positive population and a negative detection control before assigning the extranuclear color to FEN1 (HPA tissue IHC; general IHC practice). |
| Cardiomyocytes or skeletal myocytes stain strongly. | Those cells are reported as not detected in HPA tissue IHC; cross-reactivity or endogenous detection activity is possible (HPA tissue IHC; general IHC practice). | Confirm cell identity and compare antibody-omission and detection controls; reassess specificity if the unexpected cellular signal persists (general IHC practice). |
| The entire section has a diffuse brown cast. | Unresolved background can obscure the nuclear pattern needed for interpretation (HPA tissue IHC; general IHC practice). | Inspect negative controls, blocking and washing, then adjust detection or chromogen development as indicated by those controls (general IHC practice). |
| Only scattered cells stain in an otherwise weak tissue section. | HPA describes nuclear expression in highly proliferative cells rather than uniform staining of every cell in most tissues (HPA tissue IHC). | Map the positive nuclei to their cell types and compare a named High population before calling the section technically weak (HPA tissue IHC; general IHC practice). |
| An IF/ICC image appears more nucleoplasmic than nucleolar. | HPA reports nucleoplasm as the main IF/ICC location and nucleoli as an additional location (HPA subcellular ICC-IF). | Assess whether signal remains nuclear; use the separate IF/ICC guide for its workflow, and use the HPA tissue IHC pattern when scoring paraffin sections (HPA subcellular ICC-IF; HPA tissue IHC). |
Experiment evidence
IHC & IF images showing FEN1 staining
M01484-3 · IHCIHC analysis of FEN1 using anti-FEN1 antibody (M01484-3). FEN1 was detected in a paraffin-embedded section of human bladder epithelial carci
M01484-3 · IHCIHC analysis of FEN1 using anti-FEN1 antibody (M01484-3). FEN1 was detected in a paraffin-embedded section of human colorectal adenocarcinom
M01484-3 · IFIF analysis of FEN1 using anti-FEN1 antibody (M01484-3). FEN1 was detected in an immunocytochemical section of MCF-7 cells. Enzyme antigen r
M01484-4 · IHCIHC analysis of FEN1 using anti-FEN1 antibody (M01484-4). FEN1 was detected in a paraffin-embedded section of human liver cancer tissue. Hea
M01484-4 · IHCIHC analysis of FEN1 using anti-FEN1 antibody (M01484-4). FEN1 was detected in a paraffin-embedded section of human ovarian cancer tissue. H
M01484-4 · IFIF analysis of FEN1 using anti-FEN1 antibody (M01484-4). FEN1 was detected in an immunocytochemical section of MCF-7 cells. Enzyme antigen r
A01484-1 · IHCIHC analysis of FEN1 using anti-FEN1 antibody (A01484-1). FEN1 was detected in a paraffin-embedded section of human liver cancer tissue. Hea
A01484-1 · IHCIHC analysis of FEN1 using anti-FEN1 antibody (A01484-1). FEN1 was detected in a paraffin-embedded section of human ovarian serous adenocarc
A01484-1 · IFIF analysis of FEN1 using anti-FEN1 antibody (A01484-1) and anti-Beta Tubulin antibody (M01857-3). FEN1 was detected in immunocytochemical s
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000168496-FEN1/tissue/cerebral+cortex#imid_713424
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000168496-FEN1/tissue/colon#imid_713358
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000168496-FEN1/tissue/liver#imid_713367
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000168496-FEN1/tissue/kidney#imid_713360
HPA · ICC-IF U-251MGHuman Protein Atlas immunofluorescence, U-251MG, antibody CAB002262; green = FEN1, blue = nucleus, red = microtubules.
HPA · ICC-IF U-251MGHuman Protein Atlas immunofluorescence, U-251MG, antibody CAB002262; green = FEN1, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for FEN1 IHC & IF
🧪Run colon first: its glandular cells should stain for FEN1 (HPA: High in colon glandular cells). Use skeletal muscle myocytes as the negative tissue (HPA: Not detected in skeletal muscle myocytes); internal negative cells on the colon slide should show counterstain without nuclear DAB signal above background (IHC control practice).
Positive control tissue: Adrenal gland (Glandular cells, HPA High)
Negative control tissue: Heart muscle (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show FEN1 in U-251MG, U2OS, A-431, HeLa, MCF-7, U2OS, siRNA 2 (10x), U2OS, scrambled (10x), U2OS, siRNA 1 (10x), with annotated localisation: Nucleoplasm (supported) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control and a mouse IgG isotype control matched to the catalog antibody (caption: mouse anti-FEN1 antibody); confirm specificity with FEN1 knockout material or a validated peptide-block control (IHC control practice). Quench endogenous peroxidase and check the colon slide for residual DAB background (IHC control practice; caption: HRP/DAB detection).
⚠️Feasibility: A target-specific fixation window and fixation effect are unreported, and the selected SKU caption does not state a fixative (caption: M01484-3). Heat retrieval in EDTA at pH 8.0 is documented for paraffin IHC, but whether retrieval is required is unreported (caption: M01484-3). The supplied evidence does not establish frozen sections or IF as easier; in colon, assess nonspecific DAB signal with the no-primary control (IHC control practice).
HPA tissue IHC evidence for FEN1
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — High 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 |
|---|---|---|---|---|
| Adrenal gland | Glandular cells | High | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | High | Protein (IHC) | HPA → |
| Colon | Glandular cells | High | Protein (IHC) | HPA → |
| Duodenum | Glandular cells | High | Protein (IHC) | HPA → |
| Endometrium | Glandular cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Heart muscle | Cardiomyocytes | Not detected | Protein (IHC) | HPA → |
| Skeletal muscle | Myocytes | Not detected | Protein (IHC) | HPA → |
| Smooth muscle | Smooth muscle cells | Not detected | Protein (IHC) | HPA → |
| Soft tissue | Fibroblasts | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced FEN1 IHC Tips
Troubleshoot FEN1 staining in paraffin sections by checking retrieval, nuclear localisation, controls and cell specific scoring before interpreting chromogenic signal.
How should I retrieve FEN1 in paraffin sections with weak nuclear staining?
Start with heat mediated retrieval in EDTA at pH 8.0 for this catalog antibody (datasheet M01484-3). The demonstrated paraffin section workflow used that retrieval before 10% goat serum blocking and 2 μg/ml primary antibody overnight at 4°C (datasheet M01484-3). If staining remains weak, vary heating and cooling conditions on matched sections while keeping detection and exposure conditions consistent (standard IHC practice). Compare nuclear signal in proliferative areas with background in an adjacent section lacking primary antibody (HPA: nuclear expression in highly proliferative cells; standard IHC practice). Record retrieval conditions because excessive heating can damage morphology and complicate cell specific scoring (standard IHC practice).
Could fixation explain weak or uneven FEN1 staining?
Target specific fixation sensitivity is unknown: the selected paraffin section caption does not state a fixative (datasheet M01484-3). Record the actual fixative, fixation duration and processing history for each specimen before attributing weak staining to FEN1 biology (standard IHC practice). Compare similarly processed sections with the same EDTA pH 8.0 retrieval and 2 μg/ml antibody conditions used in the demonstrated workflow (datasheet M01484-3). Inspect morphology and the distribution of signal across section depth when judging uneven staining (standard IHC practice). Do not infer a FEN1 specific fixation effect from tissue staining patterns, subcellular localisation or modified residues (HPA: tissue IHC profile; UniProt P39748: localisation and modifications).
Where should convincing FEN1 chromogenic staining appear?
Prioritise staining within nuclei: tissue IHC shows nuclear expression in highly proliferative cells, and cellular imaging supports nucleoplasmic plus nucleolar localisation (HPA: tissue IHC profile; HPA: subcellular localisation). FEN1 can reside mostly in nucleoli and redistribute toward nucleoplasm after DNA damage, so record those patterns separately when morphology permits (UniProt P39748: subcellular localisation). UniProt also lists a mitochondrial pool, but diffuse cytoplasmic DAB alone does not establish that localisation in a paraffin section (UniProt P39748: subcellular localisation; standard IHC practice). Compare the signal with a nuclear counterstain and assess it within identifiable cells rather than across the whole field (standard IHC practice). If staining is mainly extracellular or confined to damaged edges, review background controls before scoring it (standard IHC practice).
Can this stain distinguish FEN1 isoforms or modified epitopes?
Do not assign chromogenic signal to a particular isoform without antibody epitope information and isoform specific validation (standard IHC practice). The record lists 2 isoforms, isoform 1 and FENMIT, but the selected tissue caption identifies no epitope or isoform selectivity (UniProt P39748: isoforms; datasheet M01484-3). FEN1 also has annotated methylation, acetylation and phosphorylation sites, including phosphoserine at residue 187 (UniProt P39748: modified residues). Those annotations do not show that this antibody detects, excludes or changes affinity for a modified form (standard IHC practice). For an isoform or modification claim, obtain epitope mapping and test an appropriate independently validated control under the same tissue processing conditions (standard IHC practice).
How can IF help assess a questionable FEN1 IHC pattern?
Use IF on a separately optimised specimen to ask whether FEN1 signal overlaps a marker for the cell population identified by morphology in the IHC section (standard IF practice). Choose fluorophores outside the strongest tissue autofluorescence, and include single channel and no primary controls before interpreting multiplex overlap (standard IF practice). HPA imaging supports nucleoplasmic and nucleolar FEN1, providing a compartment check for the chromogenic nuclear pattern (HPA: subcellular localisation). FEN1 has no annotated transmembrane segment, but the antibody epitope is unspecified, so optimise permeabilisation for nuclear access and assess mitochondrial membrane access separately if investigating that pool (UniProt P39748: topology and localisation; datasheet M01484-3: epitope unspecified). Treat IF localisation as supporting evidence rather than proof that the paraffin section stain is specific (standard IHC/IF practice).
What should I change when DAB obscures FEN1 positive nuclei?
Check a section lacking primary antibody to separate detection system background from staining associated with the primary antibody (standard IHC practice). In the demonstrated workflow, sections received 10% goat serum, 2 μg/ml primary antibody overnight at 4°C, and a peroxidase conjugated secondary before DAB development (datasheet M01484-3). Review peroxidase blocking, washing and DAB development as general chromogenic IHC steps, especially when pigment or diffuse deposits mask nuclei (standard IHC practice). Reduce primary antibody concentration or development time only through a matched titration that preserves a credible nuclear positive control (standard IHC practice; HPA: nuclear tissue IHC profile). Score identifiable nuclei after counterstaining and exclude damaged section edges from the comparison (standard IHC practice).
How should I quantify FEN1 across sections with different cell mixtures? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell compartment and region before scoring, because tissue IHC describes nuclear expression concentrated in highly proliferative cells (HPA: tissue IHC profile). Report the percentage of positive nuclei and, when intensity is reproducible, an H-score on the conventional 0–300 scale (standard IHC practice). Alternatively, report positive nuclei per mm² within a specified tissue compartment rather than across mixed tissue indiscriminately (standard IHC practice). Normalise counts to all evaluable nuclei of the same cell type in that region, and document excluded necrotic or folded areas (standard IHC practice). Keep retrieval, antibody concentration, DAB development and scoring thresholds constant across compared sections (standard IHC practice; datasheet M01484-3: retrieval and antibody conditions).
How can I distinguish true FEN1 staining from artefact?
A credible result is cell associated nuclear staining that fits the sampled cell population and survives comparison with a no primary control (HPA: nuclear tissue IHC profile; standard IHC practice). HPA reports high staining in lymph node germinal center cells and bone marrow hematopoietic cells, while cardiomyocytes and skeletal myocytes are listed as not detected (HPA: tissue IHC levels). Treat isolated extracellular deposits, section edge enhancement and necrotic staining as artefact candidates, then inspect morphology and repeat matched controls (standard IHC practice). Check peroxidase blocking when DAB appears in compartments inconsistent with FEN1 localisation (standard IHC practice; HPA: subcellular localisation). A mitochondrial annotation alone does not validate diffuse cytoplasmic chromogen as specific FEN1 staining (UniProt P39748: subcellular localisation; standard IHC practice).
Boster reagents
Best FEN1 / Flap endonuclease 1 IHC Antibodies
Three anti-FEN1 antibodies have IHC images from human paraffin sections and IF/ICC images from cultured cells (catalog image captions). Their listed reactivity covers human, mouse and, for two antibodies, rat (catalog applications/reactivity).
Real IHC data
Anti-FEN1 Antibody ® (monoclonal, 6F3F2)
Cat # M01484-3
Real IHC data
Anti-FEN1 Antibody ® (monoclonal, 7D11D7)
Cat # M01484-4
Real IHC data
Anti-FEN1 Antibody ®
Cat # A01484-1
M01484-3 has an IHC image from human bladder epithelial carcinoma, and M01484-4 has one from human liver cancer; both list IHC and IF/ICC applications and have IF images from MCF-7 cells (catalog image captions; catalog applications). A01484-1 has an IHC image from human liver cancer and IF images from U20S cells and human intestinal cancer tissue (catalog image captions).
Which to pick: For tissue IHC, choose by the matching paraffin-section example: M01484-3 for bladder epithelial carcinoma, or M01484-4 or A01484-1 for liver cancer (each SKU’s IHC image caption). For IF/ICC, all three list those applications and have corresponding images; choose the rabbit antibody A01484-1 or a mouse monoclonal, M01484-3 or M01484-4, according to the host species needed for your staining panel (catalog host, clone, applications and IF image captions). For mouse or rat work, A01484-1 and M01484-3 list both species, while M01484-4 lists mouse only; the IHC captions describe paraffin sections but do not report the fixative (catalog reactivity; each SKU’s IHC image caption).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P39748 (FEN1_HUMAN, Flap endonuclease 1).
- Human Protein Atlas. FEN1 tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. FEN1 subcellular location (ICC-IF): Mainly localized to the nucleoplasm. In addition localized to the nucleoli..
- Human Protein Atlas. FEN1 antibody validation summary (3 antibodies).
- The Clinical Significance of the Expression of FEN1 in Primary Osteosarcoma. International journal of general medicine 2021 — PMC8504935.
- Flap endonuclease 1 Facilitated Hepatocellular Carcinoma Progression by Enhancing USP7/MDM2-mediated P53 Inactivation. International journal of biological sciences 2022 — PMC8771828.
- Upregulation of FEN1 Is Associated with the Tumor Progression and Prognosis of Hepatocellular Carcinoma. Disease markers 2020 — PMC7201445.
- Overexpression and hypomethylation of flap endonuclease 1 gene in breast and other cancers. Molecular cancer research : MCR 2008 — PMC2948671.
- PubMed PMID:8007985 — UniProt-cited evidence.
- PubMed PMID:7774922 — UniProt-cited evidence.
- PubMed PMID:16554811 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for FEN1 (Flap endonuclease 1). Expected localisation Nuclear staining in tissue; nucleolar enrichment is a molecular expectation (HPA tissue IHC; UniProt). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet M01484-3). Validated IHC antibody: M01484-3 at 2 μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Adrenal gland (Glandular cells); negative Heart muscle. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human bladder epithelial carcinoma tissue; fixative not specified (datasheet M01484-3); 2. Fixation: Image fixative and duration unreported (datasheet M01484-3); 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: EDTA pH 8.0 (datasheet M01484-3); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet M01484-3); 8. Primary antibody: Mouse monoclonal (clone 6F3F2) anti-FEN1, 2 μg/ml (datasheet M01484-3); 9. Primary incubation: Overnight at 4 °C (datasheet M01484-3). Watch for: Fixation: Keep fixation consistent across sections. (standard IHC practice; not target-specific); Caveat: Cardiomyocytes may be unstained (HPA tissue IHC); Isoform / epitope: 2 isoforms, FEN1 and FENMIT; epitope coverage unknown (UniProt); Regulation: Enriched in highly proliferative cells (HPA tissue IHC). Evidence: UniProt P39748, HPA tissue IHC · Enhanced, HPA ICC-IF · Mainly localized to the nucleoplasm. In addition localized to the…, Datasheet M01484-3, PMC8504935, PMC8771828. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/FEN1/immunohistochemistry-ihc-guide.