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FBXL20 / F-box/LRR-repeat protein 20 · IHC design guide
Design Immunohistochemistry for FBXL20
Plan chromogenic FBXL20 IHC around cytoplasmic staining and cells in seminiferous ducts, where staining is high (HPA tissue IHC). Start the IHC-validated antibody at 2.5 μg/mL (datasheet), and interpret tissue patterns cautiously because their reliability is uncertain (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
FBXL20 Immunohistochemistry Experimental Design Guide
Expected localisation, validated protocols, controls and antibodies — the at-a-glance facts below, then the full design guide.
Must know before staining
| Expected localisation | Cytoplasmic in most cell types (HPA tissue IHC) | |
| Staining pattern | Cytoplasmic signal in most cell types (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen) | |
| Positive control | Testis+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 conditions consistent across sections (standard IHC practice; not target-specific) | |
| Caveat | Tissue staining is uncertain; RNA concordance is low (HPA tissue IHC) | |
| Regulation | No expression regulator annotated (UniProt) | |
| Isoform / epitope | 2 isoforms; epitope coverage is unreported (UniProt) |
Section 1
Recommended FBXL20 IHC & IF Protocols
Compare the catalog antibody’s IHC-P protocol with published FBXL20 IHC protocols for breast and pancreatic tissue (PMC8507197; PMC8570931).
| Sample | human brain tissue; fixation not specified (datasheet A12714) |
| Fixation | Image fixative and duration unreported (datasheet A12714); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat-induced epitope retrieval in citrate buffer, pH 6.0, 20 min at 95–98 °C (standard rule: cytoplasmic / membrane antigen) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% normal serum of the secondary host, 30 min, room temperature (standard) |
| Primary antibody | Rabbit anti-FBXL20, 2.5 μg/mL (datasheet A12714) |
| Primary incubation | Overnight at 4 °C (standard) |
| Detection | HRP-polymer secondary, DAB chromogen 5–10 min (standard) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | FBXL20-positive staining in cells in seminiferous ducts of testis (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression in most cell types. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6.0 retrieval at 95–98 °C for 20 min (page retrieval); the breast protocol used 98 °C for 20 min (PMC8507197).
Section 2
What Is the Expected FBXL20 Staining Pattern?
FBXL20 is cytoplasmic and has no transmembrane segment (UniProt Q96IG2 localization and topology). In paraffin sections, expect cytoplasmic staining in many cell types, especially cells in seminiferous ducts of testis, where staining is reported as high (HPA tissue IHC). Treat the pattern as provisional: HPA rates tissue IHC reliability Uncertain because antibody staining has low consistency with RNA expression data (HPA tissue IHC reliability).
| Cytoplasmic staining in cells of seminiferous ducts is stronger than nearby background. | This matches the reported high testis signal and cytoplasmic tissue profile (HPA tissue IHC). It is a useful positive-control pattern, but HPA's Uncertain reliability means morphology alone cannot establish specificity (HPA tissue IHC reliability). |
| A crisp membrane rim or predominantly nuclear stain appears without convincing cytoplasmic signal. | That compartment does not match UniProt's cytoplasmic annotation or HPA's cytoplasmic tissue profile (UniProt Q96IG2 localization; HPA tissue IHC). Review morphology and detection controls before interpreting it as FBXL20; the observed compartment may reflect nonspecific staining. |
| Strong stain appears mainly in adrenal glandular cells while the testis comparison is weak. | HPA reports adrenal glandular cells as not detected and cells in seminiferous ducts as high (HPA tissue IHC). Investigate cross-reactivity or endogenous detection activity. Because HPA tissue IHC is Uncertain, this comparison is a warning rather than proof of an unexpected biological pattern (HPA tissue IHC reliability). |
| Color spreads through tissue or extracellular spaces and cell boundaries are hard to resolve. | This is diffuse background, so a cellular FBXL20 score is unreliable. General IHC practice: inspect the no-primary control, blocking and washes, and assess whether the chromogen reaction has obscured the cytoplasm. |
| The run shows no cytoplasmic signal in cells of seminiferous ducts. | That conflicts with HPA's reported high testis staining (HPA tissue IHC). Check section integrity, retrieval and detection with appropriate run controls before calling the sample negative. HPA's Uncertain reliability limits how strongly one tissue can establish assay failure (HPA tissue IHC reliability). |
💡Expected FBXL20 appearanceCall a section positive when discernible cytoplasmic chromogen occurs in the expected cells, with high staining in cells of seminiferous ducts as the clearest supplied example (UniProt Q96IG2 localization; HPA tissue IHC); isolated membrane, nuclear or diffuse extracellular color is suspect, and the call remains provisional because HPA rates tissue IHC Uncertain (HPA tissue IHC reliability).
| Tissue and cell selection | Testis cells in seminiferous ducts are reported high; adipocytes and several listed cell populations are medium, while adrenal glandular cells are not detected (HPA tissue IHC). Choose comparison cells by their stated level rather than assuming every cell in an organ stains alike. |
| Antibody validation | Both listed antibodies have Uncertain IHC status, and the overall tissue profile awaits external verification (HPA antibodies; HPA tissue IHC reliability). A matching stain is supportive, but control agreement and cellular localization remain necessary for interpretation. |
| Topology and localization | FBXL20 has no transmembrane segment and is annotated as cytoplasmic (UniProt Q96IG2 topology and localization). Judge chromogenic staining within cell cytoplasm; a membrane-only outline is inconsistent with these supplied annotations. |
| Isoforms and epitope coverage | UniProt lists two isoforms, but the payload gives no antibody epitope or isoform coverage (UniProt Q96IG2 isoforms). Isoform-specific detection cannot be inferred from a positive or negative IHC section. |
| IF/ICC: should a filament-like pattern be expected? | HPA reports approved microtubule localization by ICC-IF, with images from Rh30, SiHa and U2OS (HPA subcellular ICC-IF). This supports an IF interpretation question; it does not establish that paraffin chromogenic IHC must resolve individual microtubules (HPA tissue IHC). |
| Situation | Likely cause | Next action |
|---|---|---|
| Testis control is blank. | The expected high signal was not observed in cells of seminiferous ducts (HPA tissue IHC); the cause is undetermined. | General IHC practice: check section quality, retrieval, primary incubation, detection reagents and a working run control. Do not assign a FBXL20-specific fixation sensitivity; none is supplied. |
| Most cells stain uniformly, including a no-primary control. | A detection-system contribution is possible; uniform color does not demonstrate the HPA cytoplasmic cell pattern (HPA tissue IHC). | General IHC practice: inspect endogenous enzyme or biotin controls as relevant to the detection system, adjust blocking and washes, and compare with a no-primary section. |
| Only nuclei or cell borders stain. | The observed compartment conflicts with the cytoplasmic annotation and lacks the reported cytoplasmic tissue pattern (UniProt Q96IG2 localization; HPA tissue IHC). | General IHC practice: confirm cell boundaries with the counterstain, inspect control sections and reassess background before scoring the stain as FBXL20. |
| Adrenal glandular cells are strong while testis cells are weak. | This reverses the supplied not-detected versus high comparison, though the HPA IHC profile itself is Uncertain (HPA tissue IHC). | Compare matched runs and no-primary controls; investigate cross-reactivity or endogenous detection activity before claiming a tissue-specific exception. |
| Cytoplasmic color is too diffuse to score individual cells. | Background or excessive chromogen development may obscure the reported cellular pattern (HPA tissue IHC profile; general IHC practice). | General IHC practice: inspect the no-primary control, review blocking and washing, and shorten chromogen development if the run conditions support that adjustment. |
| IF/ICC shows microtubule-associated signal, but paraffin IHC looks broadly cytoplasmic. | HPA describes microtubules in ICC-IF and cytoplasmic expression in tissue IHC (HPA subcellular ICC-IF; HPA tissue IHC). The two observations have different reported resolution. | Interpret each assay against its own HPA annotation. Assess the IHC section for cellular cytoplasmic staining and controls; use the separate IF/ICC guide for imaging workflow. |
Experiment evidence
IHC & IF images showing FBXL20 staining
A12714 · IHCImmunohistochemical staining of human brain tissue using Scrapper antibody at 2.5 μg/mL.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000108306-FBXL20/tissue/cerebral+cortex#imid_15915876
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000108306-FBXL20/tissue/colon#imid_15939247
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000108306-FBXL20/tissue/liver#imid_15939147
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000108306-FBXL20/tissue/kidney#imid_15939235
HPA · ICC-IF Rh30Human Protein Atlas immunofluorescence, Rh30, antibody HPA050397; green = FBXL20, blue = nucleus, red = microtubules.
HPA · ICC-IF Rh30Human Protein Atlas immunofluorescence, Rh30, antibody HPA050397; green = FBXL20, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for FBXL20 IHC & IF
🧪Run testis first and expect staining in cells of the seminiferous ducts (HPA: High); run adrenal gland as the negative tissue and expect glandular cells to remain at background (HPA: Not detected). On the testis slide, cells without specific signal should remain at background, but the supplied HPA rows do not identify an internal negative cell type (HPA: testis row).
Positive control tissue: Testis (Cells in seminiferous ducts, HPA High)
Negative control tissue: Adrenal gland (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show FBXL20 in Rh30, SiHa, U2OS, with annotated localisation: Microtubules (approved) (HPA subcellular).
Technical controls: Include a no-primary (secondary-only) control, plus an isotype control matched to the primary antibody’s host species and clonality where applicable; use knockout tissue or peptide competition, if available, as a biological specificity control (standard IHC practice). For chromogenic staining of testis, quench endogenous peroxidase and check background in the no-primary control (standard IHC practice).
⚠️Feasibility: A target-specific fixation window, fixation effect, and antigen-retrieval dependency are unreported in the supplied evidence; the fixative in the A12714 tissue-IHC caption is also unreported (A12714 caption). Optimize retrieval empirically for paraffin sections (standard IHC practice). IF/ICC images support examining FBXL20 localisation at microtubules, but the supplied evidence does not establish that IF or frozen sections are easier than paraffin IHC (HPA: approved microtubule localisation; HPA: testis High).
HPA tissue IHC evidence for FBXL20
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Uncertain — Low consistency between antibody staining and RNA expression data. Pending external verification.). 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 |
|---|---|---|---|---|
| Testis | Cells in seminiferous ducts | High | Protein (IHC) | HPA → |
| Adipose tissue | Adipocytes | Medium | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | Medium | Protein (IHC) | HPA → |
| Breast | Adipocytes | Medium | Protein (IHC) | HPA → |
| Endometrium | Glandular cells | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adrenal gland | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Caudate | Glial cells | Not detected | Protein (IHC) | HPA → |
| Cerebellum | Cells in granular layer | Not detected | Protein (IHC) | HPA → |
| Cervix | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Duodenum | Glandular cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced FBXL20 IHC Tips
Troubleshoot FBXL20 chromogenic IHC by checking retrieval, tissue preservation, cytoplasmic localisation and controls before interpreting staining intensity (UniProt Q96IG2; standard IHC practice).
How should I adjust retrieval when FBXL20 staining is weak in paraffin sections?
Use citrate buffer at pH 6.0 for heat-induced retrieval at 95–98 °C for 20 min before chromogenic IHC (page retrieval specification). This is a starting condition; the catalog brain-tissue image reports 2.5 µg/mL antibody but does not report retrieval conditions (caption A12714). If cytoplasmic staining remains weak, compare heating times on adjacent sections, then test EDTA at pH 9.0 as a fallback (standard IHC optimization). Hold antibody concentration and DAB development time constant while comparing signal, background and tissue morphology (standard IHC practice).
Could fixation explain inconsistent FBXL20 staining between tissue blocks?
Target-specific fixation sensitivity is unknown: the selected brain-tissue caption gives antibody concentration but no fixative or fixation time (caption A12714). For new specimens, record tissue thickness and time in 10% neutral buffered formalin, then compare otherwise matched blocks fixed for 6 and 24 h as an optimization exercise (standard IHC practice). Process and retrieve those blocks together so fixation is the principal variable in the comparison (standard IHC practice). Assess preservation of morphology alongside cytoplasmic DAB signal, because damaged tissue can make a stronger stain less interpretable (standard IHC practice; UniProt Q96IG2 localisation).
Should FBXL20 appear diffuse in cytoplasm or follow microtubules?
Expect a cytoplasmic IHC signal as the broad localisation reference (UniProt Q96IG2; HPA tissue-IHC profile: cytoplasmic expression in most cell types). HPA also reports an approved microtubule location from subcellular imaging, but fine filament detail may be difficult to resolve with chromogenic staining in paraffin sections (HPA subcellular; standard IHC practice). Compare the pattern within intact cells rather than treating a crisp nuclear-only deposit as expected FBXL20 localisation (UniProt Q96IG2; standard IHC interpretation). Include a tissue control across runs, and document whether staining is diffuse cytoplasmic, filament-like or mixed before drawing a localisation conclusion (standard IHC practice).
Can epitope choice explain different FBXL20 IHC patterns?
FBXL20 has 2 annotated isoforms, but the supplied evidence does not map this antibody’s epitope or establish which isoforms it recognises (UniProt Q96IG2; caption A12714). The F-box spans residues 22–68, and annotated phosphorylation sites include residues 417 and 421; these features alone do not establish retrieval sensitivity (UniProt Q96IG2). If antibody documentation later provides an immunogen sequence, map it against both isoforms before attributing staining differences to splicing (standard epitope assessment). Compare an independently validated epitope and appropriate controls on adjacent sections when an isoform-specific interpretation matters (standard IHC validation practice).
How can IF help assess an ambiguous FBXL20 IHC pattern?
Use IF as a separate localisation check, pairing FBXL20 with a validated marker for the cell population being assessed, such as cells in seminiferous ducts (HPA tissue IHC: high in those cells; standard IF practice). Choose a far-red fluorophore when tissue autofluorescence compromises shorter wavelengths, and inspect single-stain and unstained controls before interpreting overlap (standard IF practice). FBXL20 is cytoplasmic and has no transmembrane segment, so allow intracellular antibody access with a mild permeabilisation trial, such as 0.1% detergent for 5 min (UniProt Q96IG2 topology; standard IF practice). Compare IF localisation with the IHC pattern cautiously because HPA tissue-IHC reliability is uncertain (HPA tissue IHC).
How do I separate FBXL20 signal from chromogenic background?
Inspect a section processed without primary antibody to reveal detection-related deposits before assigning DAB staining to FBXL20 (standard IHC control practice). Block endogenous peroxidase, for example with 3% hydrogen peroxide for 10 min, and apply an appropriate protein block before the primary antibody (standard chromogenic IHC practice). If background persists, compare shorter DAB development and a lower primary concentration while keeping retrieval and tissue processing matched (standard IHC optimization). Judge improvement by cytoplasmic contrast in intact cells, since that compartment agrees with the supplied FBXL20 localisation evidence (UniProt Q96IG2; HPA tissue-IHC profile).
How should I score FBXL20 staining across heterogeneous sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and viable analysis area before scoring cytoplasmic DAB, and apply the same thresholds to every section (UniProt Q96IG2 localisation; standard IHC quantification practice). Record the percentage of positive cells and intensity from 0–3; an H-score sums each intensity multiplied by its cell percentage, yielding 0–300 (standard IHC scoring practice). Alternatively, report positive cells per mm², normalised to viable tissue area or the counted cells in the prespecified population (standard image-analysis practice). Keep staining batches and imaging settings comparable, and report background-control results because HPA rates its tissue-IHC evidence uncertain (standard IHC practice; HPA tissue IHC).
What findings support genuine FBXL20 staining rather than artefact?
A reproducible intracellular cytoplasmic pattern is consistent with FBXL20 localisation, while nuclear-only deposits require further validation (UniProt Q96IG2; HPA tissue-IHC profile; standard IHC interpretation). Check the stained cell population against the section’s morphology; HPA reports high staining in cells of seminiferous ducts but rates overall tissue-IHC reliability uncertain (HPA tissue IHC). Exclude necrotic regions and section edges when judging intensity, and compare primary-omission controls for endogenous enzyme or detection deposits (standard IHC practice). The catalog brain-tissue caption documents staining at 2.5 µg/mL but supplies neither fixation conditions nor a scored cellular pattern for independent confirmation (caption A12714).
Boster reagents
Best FBXL20 / F-box/LRR-repeat protein 20 IHC Antibodies
A12714 has an IHC image from human brain tissue at 2.5 μg/mL (catalog image caption). Human, mouse, and rat reactivity is listed; no IF data are supplied (catalog: A12714).
Real IHC data
Anti-SCRAPPER FBXL20 Antibody
Cat # A12714
A12714 is the only card and lists IHC-P (catalog: A12714); its image shows staining of human brain tissue at 2.5 μg/mL (catalog image caption). Human, mouse, and rat reactivity is listed, while IF/ICC validation is unreported (catalog: A12714).
Which to pick: Choose A12714 for paraffin-section IHC (catalog: IHC-P); its IHC image depicts human brain tissue, but the fixative is unreported (catalog image caption). There is no IF/ICC pick because A12714 has no listed IF/ICC application or image (catalog: A12714). For cross-species planning, A12714 lists human, mouse, and rat reactivity, though its depicted IHC evidence is limited to human brain tissue (catalog: A12714; catalog image caption).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q96IG2 (FXL20_HUMAN, F-box/LRR-repeat protein 20).
- Human Protein Atlas. FBXL20 tissue IHC expression (reliability: Uncertain).
- Human Protein Atlas. FBXL20 subcellular location (ICC-IF): Localized to the microtubules..
- Human Protein Atlas. FBXL20 antibody validation summary (2 antibodies).
- FBXL20 promotes breast cancer malignancy by inhibiting apoptosis through degradation of PUMA and BAX. The Journal of biological chemistry 2021 — PMC8507197.
- p53/FBXL20 axis negatively regulates the protein stability of PR55α, a regulatory subunit of PP2A Ser/Thr phosphatase. Neoplasia (New York, N.Y.) 2021 — PMC8570931.
- Age-related copy number variations and expression levels of F-box protein FBXL20 predict ovarian cancer prognosis. Translational oncology 2020 — PMC7486480.
- PubMed PMID:14702039 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
- PubMed PMID:18088087 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for FBXL20 (F-box/LRR-repeat protein 20). Expected localisation Cytoplasmic in most cell types (HPA tissue IHC). Antigen retrieval: Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen). Validated IHC antibody: A12714 at 2.5 μg/mL (the product's own IHC validation image is shown). Controls (HPA): positive Testis (Cells in seminiferous ducts); negative Adrenal gland. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: human brain tissue; fixation not specified (datasheet A12714); 2. Fixation: Image fixative and duration unreported (datasheet A12714); verify before use.; 3. Sectioning: 4–5 µm sections on charged slides (standard); 4. Deparaffinisation: Xylene, graded ethanol series to water (standard); 5. Antigen retrieval: Heat-induced epitope retrieval in citrate buffer, pH 6.0 , 20 min at 95–98 °C…; 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% normal serum of the secondary host, 30 min , room temperature (standard); 8. Primary antibody: Rabbit anti-FBXL20, 2.5 μg/mL (datasheet A12714); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation conditions consistent across sections (standard IHC practice; not target-specific); Caveat: Tissue staining is uncertain; RNA concordance is low (HPA tissue IHC); Isoform / epitope: 2 isoforms; epitope coverage is unreported (UniProt); Regulation: No expression regulator annotated (UniProt). Evidence: UniProt Q96IG2, HPA tissue IHC · Uncertain, HPA ICC-IF · microtubules., Datasheet A12714, PMC8507197, PMC8570931. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/FBXL20/immunohistochemistry-ihc-guide.