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FHL1 / Four and a half LIM domains protein 1 · IHC design guide
Design Immunohistochemistry for FHL1
Plan FHL1 staining in paraffin sections using skeletal muscle myocytes or thyroid glandular cells as positive controls (HPA tissue IHC). The catalog antibody’s IHC starting concentration is 0.5–1 μg/mL (datasheet A01258-1); assess staining by cell type because tissue compartments remain unconfirmed (HPA tissue IHC; UniProt).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
FHL1 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 | Cytoplasm or nucleus; tissue compartment unconfirmed (UniProt) | |
| Staining pattern | Strong myocyte and thyroid glandular-cell staining; compartment unreported (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A01258-1) | |
| Positive control | Endometrium+4 more · see all | |
| Negative control | Adrenal gland+4 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent across paraffin sections. (standard IHC practice; not target-specific) | |
| Caveat | Cell-type differences can complicate tissue-level scoring (HPA tissue IHC) | |
| Regulation | Isoform 1 is enriched in skeletal muscle (UniProt) | |
| Isoform / epitope | Five isoforms; epitope coverage is undetermined (UniProt) |
Section 1
Recommended FHL1 IHC & IF Protocols
The catalog antibody has an IHC-P protocol (datasheet A01258-1); the published options below describe FHL1 staining in glioma, thyroid carcinoma, and human myocardial tissue (PMC7202308; PMC11551809; PMC6687666).
| Sample | Paraffin-embedded human rectal cancer tissue; fixative not specified (datasheet A01258-1) |
| Fixation | Image fixative and duration unreported (datasheet A01258-1); 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 A01258-1); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A01258-1) |
| Primary antibody | Rabbit anti-FHL1, 0.5-1μg/ml (datasheet A01258-1) |
| Primary incubation | Overnight at 4 °C (datasheet A01258-1) |
| Detection | Streptavidin-biotin complex (SABC), DAB chromogen (datasheet A01258-1) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | FHL1-positive staining in smooth muscle cells of endometrium (HPA tissue IHC: High). HPA tissue profile: Distinct expression in muscle tissue and thyroid glands. No signal in the no-primary control. |
💡Decision noteStart with EDTA pH 8.0 heat retrieval (datasheet A01258-1). If optimizing, a published glioma protocol used citrate pH 6 retrieval (PMC7202308).
Section 2
What Is the Expected FHL1 Staining Pattern?
FHL1 staining is expected mainly in the cytoplasm, with nuclear localisation possible in some cell states (UniProt Q13642: subcellular location). In paraffin sections, skeletal myocytes are a strong positive reference; smooth muscle cells and thyroid glandular cells are also reported as high staining (HPA: High in these cells). FHL1 has no transmembrane segment (UniProt Q13642: topology). HPA rates the tissue IHC profile Enhanced, with medium consistency between staining and RNA expression (HPA: reliability).
| Strong staining in skeletal myocytes, with signal mainly in the cell body. | This fits the high skeletal-myocyte staining reported by HPA and FHL1's cytoplasmic localisation (HPA: High in skeletal myocytes; UniProt Q13642: cytoplasm). Assess the muscle cells themselves: a dark field without identifiable positive cells is insufficient to establish the expected pattern (general IHC interpretation). |
| Signal appears exclusively in nuclei, or as a sharp rim around many unrelated cells. | Review the cell type and controls before calling this positive. Nuclear FHL1 is possible, especially in myoblasts (UniProt Q13642: subcellular location), and plasma-membrane signal is reported in ICC-IF (HPA: supported additional location). Neither source establishes a universal nuclear-only or membrane-rim pattern for paraffin tissue. |
| Strong colour appears in cell types expected to be unstained, including bronchial respiratory epithelium. | The reported lack of staining in that cell type makes cross-reactivity or endogenous detection activity plausible explanations, subject to controls (HPA: Not detected in bronchial respiratory epithelial cells; general IHC practice). Identify the stained cells before treating nearby positive structures as evidence for epithelial FHL1. |
| Colour spreads across cell-free areas or obscures boundaries between cells. | This prevents a reliable compartment or cell-type call (general IHC interpretation). A diffuse deposit is compatible with excess reagent, incomplete blocking or detection background (general IHC practice); it does not reproduce the cell-resolved high staining reported for skeletal myocytes (HPA: High in skeletal myocytes). |
| No convincing signal appears in a skeletal-muscle section. | This conflicts with the reported high staining in skeletal myocytes (HPA: High in skeletal myocytes), but one negative section cannot establish absent FHL1. Check tissue preservation and the staining run with appropriate controls (general IHC practice). The HPA tissue profile has only medium consistency with RNA data (HPA: reliability description). |
💡Expected FHL1 appearanceA convincing positive shows high, cell-resolved staining in skeletal myocytes, usually cytoplasmic; colour limited to cell-free areas or unexpected cell types warrants control review (HPA: High in skeletal myocytes; UniProt Q13642: cytoplasm; general IHC interpretation).
| Cell state and compartment | FHL1 is predominantly nuclear in myoblasts but cytosolic in differentiated myotubes (UniProt Q13642: subcellular location). Interpret nuclear signal with cell identity and controls; the myoblast observation does not define a paraffin-section scoring rule. |
| Tissue and cell identity | HPA reports high staining in skeletal myocytes, endometrial smooth muscle cells and thyroid glandular cells, but no detection in bronchial respiratory epithelial cells (HPA: tissue IHC). These are cell-specific references; do not score an entire organ as uniformly positive or negative. |
| Isoform distribution | Isoform 1 is highly expressed in skeletal muscle; isoforms 2 and 3 have different reported tissue distributions (UniProt Q13642: tissue specificity). The supplied record gives no antibody epitope or isoform coverage, so staining cannot identify a particular isoform. |
| Evidence strength | The tissue IHC profile is Enhanced, while its consistency with RNA expression is medium (HPA: reliability). Three listed antibodies have Enhanced IHC status (HPA: HPA001040, HPA001391, CAB020817); this supports the observed profile without proving every new reagent or section will match it. |
| Topology and processing | FHL1 has no transmembrane segment or signal peptide, and the listed protein chain begins at residue 2 (UniProt Q13642: topology and processing). These annotations offer no basis to expect a secreted extracellular stain or to infer a fixation-sensitive epitope. |
| Situation | Likely cause | Next action |
|---|---|---|
| Skeletal myocytes remain unstained. | A weak run, unsuitable working dilution or ineffective antigen retrieval may reduce IHC signal (general IHC practice); high staining is reported in these cells (HPA: skeletal myocytes). | Check a known-positive section and run controls, then optimise retrieval and antibody dilution within the reagent's validated IHC workflow (general IHC practice). The supplied sources give no FHL1-specific retrieval condition or fixation sensitivity. |
| The whole section looks brown or cell borders cannot be resolved. | Residual detection activity, incomplete blocking or overly concentrated reagents can create broad background (general IHC practice). | Inspect the no-primary control, review blocking and washes, and titrate primary or detection reagents (general IHC practice). Reassess whether staining resolves to skeletal myocytes, where HPA reports high signal (HPA: skeletal myocytes). |
| Bronchial respiratory epithelium stains strongly. | This differs from the HPA observation of no detection in those epithelial cells; cross-reactivity or endogenous activity is possible (HPA: bronchus; general IHC practice). | Confirm cell identity and compare no-primary and detection controls (general IHC practice). If staining persists only with primary antibody, assess reagent specificity before reporting epithelial FHL1; HPA's negative observation is a reference, not proof of universal absence. |
| Only nuclei stain in the putative positive cells. | Nuclear FHL1 can occur in myoblasts, while differentiated myotubes are described as cytosolic (UniProt Q13642: subcellular location). Tissue cell state may be uncertain. | Verify the stained cell population and inspect cytoplasmic signal in a positive reference (HPA: High in skeletal myocytes; general IHC practice). Do not reject every nuclear signal solely on compartment, or accept nuclear-only staining without controls. |
| High staining is patchy across a tissue section. | The HPA entries refer to named cell types, including endometrial smooth muscle cells and thyroid glandular cells (HPA: tissue IHC); their distribution need not fill the section. | Score the specified cells separately from neighbours and document their location and intensity (general IHC interpretation). Compare with an appropriate positive reference; avoid treating a whole-tissue average as the reported HPA cell-level result. |
| IF/ICC Q&A: should cytosolic or membrane signal be expected? | HPA calls cytosol the main location with uncertain support and plasma membrane an additional location with supported evidence (HPA: subcellular ICC-IF). UniProt also records nuclear localisation in myoblasts (UniProt Q13642: subcellular location). | Interpret IF/ICC with its own cell-state and compartment controls (general IF practice). The ICC-IF observations can inform localisation, but do not establish a paraffin IHC pattern or supply an IF/ICC protocol. |
Experiment evidence
IHC & IF images showing FHL1 staining
A01258-1 · IHCIHC analysis of FHL using anti-FHL antibody (A01258-1). FHL was detected in paraffin-embedded section of human rectal cancer tissue. Heat me
A01258-1 · IHCIHC analysis of FHL using anti-FHL antibody (A01258-1). FHL was detected in paraffin-embedded section of human renal cancer tissue. Heat med
A01258-1 · IFIF analysis of FHL using anti-FHL antibody (A01258-1). FHL was detected in immunocytochemical section of U20S cells. Enzyme antigen retrieva
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000022267-FHL1/tissue/cerebral+cortex#imid_5421196
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000022267-FHL1/tissue/colon#imid_5421106
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000022267-FHL1/tissue/liver#imid_5421107
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000022267-FHL1/tissue/kidney#imid_5421116
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA001040; green = FHL1, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA001040; green = FHL1, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for FHL1 IHC & IF
🧪Run skeletal muscle first; myocytes should stain strongly (HPA: High in skeletal-muscle myocytes). Use adrenal gland glandular cells as the negative tissue (HPA: Not detected); on the skeletal-muscle slide, any unstained nonmyocyte cells should show only background chromogen, but their FHL1 status is unreported (HPA: skeletal-muscle myocytes High).
Positive control tissue: Endometrium (Smooth muscle cells, HPA High)
Negative control tissue: Adrenal gland (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show FHL1 in A-431, U-251MG, U2OS, with annotated localisation: Cytosol (uncertain) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control, a concentration-matched rabbit IgG isotype control, and FHL1 knockout tissue if available (selected-SKU caption: rabbit primary antibody). For chromogenic detection, quench endogenous peroxidase and check endogenous biotin background with the biotin-based detection system (selected-SKU caption: biotinylated secondary, streptavidin–biotin complex and DAB).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected-SKU paraffin-section caption does not state a fixative. That caption reports heat-mediated EDTA retrieval at pH 8.0, but does not establish how strongly detection depends on retrieval (selected-SKU caption). There is no matched evidence that frozen sections or IF/ICC are easier; in skeletal muscle, compare any fiber-edge chromogen with the negative controls before scoring it as specific staining (HPA: High in skeletal-muscle myocytes).
HPA tissue IHC evidence for FHL1
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 |
|---|---|---|---|---|
| Endometrium | Smooth muscle cells | High | Protein (IHC) | HPA → |
| Lung | Alveolar cells type I | High | Protein (IHC) | HPA → |
| Placenta | Syncytiotrophoblasts - cell body | High | Protein (IHC) | HPA → |
| Skeletal muscle | Myocytes | High | Protein (IHC) | HPA → |
| Testis | Elongated or late spermatids | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adrenal gland | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Appendix | Endocrine 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 FHL1 IHC Tips
Use cell type, compartment, and matched controls to troubleshoot FHL1 staining in paraffin section IHC (HPA tissue IHC; UniProt Q13642).
What should I change when FHL1 staining is weak after retrieval?
Start with heat-mediated retrieval in EDTA at pH 8.0 for this paraffin-section assay (datasheet A01258-1). The selected image used that retrieval before incubation with 1 μg/ml antibody overnight at 4°C, so verify those recorded conditions before changing retrieval (caption A01258-1). Compare a well-stained control section and the weak section in the same run, keeping detection and DAB development matched (standard IHC practice). If signal remains weak, test a second retrieval condition on serial sections as a fallback and check whether it increases background or damages morphology (standard IHC practice). Do not interpret absent staining from retrieval alone: FHL1 expression varies by tissue and cell type (UniProt Q13642; HPA tissue IHC).
Can fixation explain weak or uneven FHL1 staining in paraffin sections?
Target-specific fixation sensitivity for FHL1 is unknown from the supplied evidence; the selected image identifies a paraffin section but does not state its fixative (caption A01258-1). Record the actual fixative, fixation duration, processing history, and section age for each specimen before comparing staining (standard IHC practice). Run specimens with different histories alongside the same control tissue using matched EDTA pH 8.0 retrieval and detection (datasheet A01258-1; standard IHC practice). Assess morphology and within-section staining consistency before attributing a weak result to fixation (standard IHC practice). Neither tissue expression patterns nor FHL1 topology establishes how its epitope responds to fixation (HPA tissue IHC; UniProt Q13642).
Should FHL1 stain nuclei, cytoplasm, or cell borders in IHC?
Evaluate the stained cell and its compartment together: FHL1 is reported in cytoplasm and nucleus, with predominantly nuclear localisation in myoblasts and cytosolic localisation in differentiated myotubes (UniProt Q13642). HPA subcellular data describe a mainly cytosolic pattern with uncertain confidence and an additional supported plasma-membrane localisation (HPA subcellular). FHL1 has no transmembrane segment, so a crisp border-only pattern warrants comparison with adjacent cells and a matched negative control (UniProt Q13642 topology; standard IHC practice). For paraffin sections, inspect nuclei and cytoplasm at high magnification while checking that the chromogen follows intact cell boundaries rather than folds or section edges (standard IHC practice).
Could an unrecognised FHL1 isoform account for missing staining?
FHL1 has 5 listed isoforms, and their tissue distributions differ, so a negative region need not represent every isoform equally (UniProt Q13642). Isoform 1 is highly expressed in skeletal muscle; isoform 2 is reported in brain and skeletal muscle; isoform 3 is reported in testis, heart, and skeletal muscle (UniProt Q13642). The supplied caption does not map this antibody’s epitope or establish which isoforms it detects (caption A01258-1). Check its documented immunogen or epitope information before making an isoform-specific claim, then compare staining in an appropriate positive cell population under matched retrieval and detection conditions (standard IHC practice; HPA tissue IHC).
How should I assess FHL1 by IF alongside this IHC assay?
Use IF as a separate assay and multiplex FHL1 with a marker identifying the expected cell population, such as myocytes in skeletal muscle or smooth muscle cells in endometrium (HPA tissue IHC). Choose fluorophores and filter settings after checking specimen autofluorescence, and include single-label controls to assess bleed-through (standard IF practice). FHL1 has no transmembrane segment and has reported intracellular localisation; use permeabilisation when assessing a cytosolic or nuclear epitope, while recognising that this antibody’s epitope is not mapped here (UniProt Q13642; caption A01258-1; standard IF practice). Compare compartment patterns cautiously: HPA reports mainly cytosolic IF localisation with uncertain confidence and additional supported plasma-membrane localisation (HPA subcellular).
How can I identify nonspecific brown signal in FHL1 IHC?
The selected assay uses a biotinylated secondary, streptavidin–biotin complex, and DAB, making reagent-only and primary-omission controls useful for tracing unwanted colour (caption A01258-1; standard IHC practice). Include a peroxidase block in a chromogenic workflow and evaluate endogenous biotin interference when using this detection chemistry (standard IHC practice). Check whether signal persists without primary antibody, collects at tissue edges, or coats damaged areas instead of outlining intact cells (standard IHC practice). The selected section was blocked with 10% goat serum; compare matched blocking and washing conditions before increasing antibody concentration (caption A01258-1; standard IHC practice). Interpret diffuse colour against a cell-type control rather than treating every brown deposit as FHL1 (HPA tissue IHC; standard IHC practice).
How should I score FHL1 staining across tissue sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring, because FHL1 can be cytoplasmic or nuclear and tissue staining varies by cell type (UniProt Q13642; HPA tissue IHC). Record the percentage of positive target cells and staining intensity, or calculate an H-score from intensity categories across those cells (standard IHC practice). Normalise the result to the number of eligible target cells or their annotated tissue area, rather than total section area when cell composition differs (standard IHC practice). Keep retrieval, antibody incubation, DAB development, imaging, and scoring thresholds matched across comparisons (datasheet A01258-1; caption A01258-1; standard IHC practice). Report nuclear and cytoplasmic scores separately when both compartments are evaluated (UniProt Q13642; standard IHC practice).
What evidence separates genuine FHL1 staining from artefact?
Look for reproducible staining in intact expected cells: HPA reports high FHL1 staining in skeletal-muscle myocytes and endometrial smooth muscle cells (HPA tissue IHC). Check whether the signal occupies plausible cytoplasmic or nuclear compartments, while allowing that compartment distribution can depend on differentiation state (UniProt Q13642). Treat staining concentrated at section edges, folds, or necrotic areas as suspect until it survives review of adjacent intact tissue and controls (standard IHC practice). With DAB and streptavidin–biotin detection, use primary-omission and detection controls to investigate endogenous enzyme or biotin-related colour (caption A01258-1; standard IHC practice). HPA labels its tissue IHC reliability “Enhanced” while describing medium consistency with RNA data, so assess discordant cells individually (HPA tissue IHC).
Boster reagents
Best FHL1 / Four and a half LIM domains protein 1 IHC Antibodies
The catalog lists two anti-FHL1 antibodies for IHC; A01258-1 also has human paraffin-section IHC and cell IF images (catalog applications/reactivity; A01258-1 image captions).
Real IHC data
Anti-FHL FHL1 Antibody ®
Cat # A01258-1
A01258-1 has IHC images from human rectal and renal cancer paraffin sections and an IF image from U20S cells (A01258-1 image captions). M01258 lists IHC as an application and human, mouse and rat reactivity, but has no supplied IHC image (catalog applications/reactivity; M01258 image captions).
Which to pick: For tissue IHC, choose A01258-1 when a documented human paraffin-section example is useful; its caption reports EDTA retrieval at pH 8.0, but does not report the fixative (A01258-1 IHC image captions). For IF/ICC, choose A01258-1 because both applications are listed and its IF caption documents U20S cells (catalog applications; A01258-1 IF image caption). For mouse or rat IHC, M01258 is the catalog-listed option based on its stated reactivity and IHC application; it is monoclonal, and no sample image is supplied (catalog host/clone/applications/reactivity; M01258 image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q13642 (FHL1_HUMAN, Four and a half LIM domains protein 1).
- Human Protein Atlas. FHL1 tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. FHL1 subcellular location (ICC-IF): Mainly localized to the cytosol. In addition localized to the plasma membrane..
- Human Protein Atlas. FHL1 antibody validation summary (3 antibodies).
- Downregulation of FHL1 protein in glioma inhibits tumor growth through PI3K/AKT signaling. Oncology letters 2020 — PMC7202308.
- FHL1: A novel diagnostic marker for papillary thyroid carcinoma. Pathology international 2024 — PMC11551809.
- Transcriptional Analysis Reveals That the FHL1/JAK-STAT Pathway is Involved in Acute Cartilage Injury in Mice. Cartilage 2025 — PMC11948231.
- Molecular autopsy and family screening in a young case of sudden cardiac death reveals an unusually severe case of FHL1 related hypertrophic cardiomyopathy. Molecular genetics & genomic medicine 2019 — PMC6687666.
- PubMed PMID:8753811 — UniProt-cited evidence.
- PubMed PMID:9714789 — UniProt-cited evidence.
- PubMed PMID:10352231 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for FHL1 (Four and a half LIM domains protein 1). Expected localisation Cytoplasm or nucleus; tissue compartment unconfirmed (UniProt). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A01258-1). Validated IHC antibody: A01258-1 at 0.5-1μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Endometrium (Smooth muscle cells); negative Adrenal gland. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human rectal cancer tissue; fixative not specified (datasheet A01258-1); 2. Fixation: Image fixative and duration unreported (datasheet A01258-1); 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 retrieval: EDTA pH 8.0 (datasheet A01258-1); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A01258-1); 8. Primary antibody: Rabbit anti-FHL1, 0.5-1μg/ml (datasheet A01258-1); 9. Primary incubation: Overnight at 4 °C (datasheet A01258-1). Watch for: Fixation: Keep fixation consistent across paraffin sections. (standard IHC practice; not target-specific); Caveat: Cell-type differences can complicate tissue-level scoring (HPA tissue IHC); Isoform / epitope: Five isoforms; epitope coverage is undetermined (UniProt); Regulation: Isoform 1 is enriched in skeletal muscle (UniProt). Evidence: UniProt Q13642, HPA tissue IHC · Enhanced, HPA ICC-IF · Mainly localized to the cytosol. In addition localized to the plasma…, Datasheet A01258-1, PMC7202308, PMC11551809. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/FHL1/immunohistochemistry-ihc-guide.