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IFT20 / Intraflagellar transport protein 20 homolog · IHC design guide
Design Immunohistochemistry for IFT20
Plan chromogenic IHC for IFT20 around its cytoplasmic tissue staining (HPA tissue IHC). Compare high-staining appendix glandular cells with low-staining liver cholangiocytes (HPA tissue IHC), and start within the catalog antibody’s 2–5 μg/ml IHC range (datasheet A07253).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
IFT20 Immunohistochemistry Experimental Design Guide
Expected localisation, validated protocols, controls and antibodies — the at-a-glance facts below, then the full design guide.
Must know before staining
| Expected localisation | Cytoplasmic tissue staining (HPA tissue IHC) | |
| Staining pattern | Cytoplasmic staining across tissues (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A07253) | |
| Positive control | Appendix+4 more · see all | |
| Negative control | None in HPA (detected in all 45 tissues); use no-primary + isotype controls |
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 and RNA show medium consistency (HPA tissue IHC) | |
| Regulation | Low tissue specificity (HPA tissue RNA) | |
| Isoform / epitope | Four isoforms; epitope coverage is unreported (UniProt) |
Section 1
Recommended IFT20 IHC & IF Protocols
The catalog antibody protocol uses heat-mediated EDTA pH 8.0 retrieval (datasheet A07253). One published IHC protocol provides a comparison in human lung tissue (PMC9308367).
| Sample | Paraffin-embedded human colon cancer tissue; fixative not specified (datasheet A07253) |
| Fixation | Image fixative and duration unreported (datasheet A07253); 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 A07253); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A07253) |
| Primary antibody | Rabbit anti-IFT20, 2-5 μg/ml (datasheet A07253) |
| Primary incubation | Overnight at 4 °C (datasheet A07253) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet A07253) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | IFT20-positive staining in glandular cells of appendix (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression in all tissues. No signal in the no-primary control. |
💡Decision noteStart with EDTA pH 8.0 retrieval for the catalog antibody (datasheet A07253). The published high-temperature, high-pressure retrieval lasts 1–2 min; its buffer is unspecified (PMC9308367).
Section 2
What Is the Expected IFT20 Staining Pattern?
IFT20 is a cytoplasmic protein associated with the Golgi, centrosomes, basal bodies and cilia (UniProt Q8IY31 localization; no transmembrane segment). In paraffin-section IHC, expect cytoplasmic staining across many tissues, including strong staining in several glandular and epithelial cell groups (HPA: cytoplasmic expression in all tissues; selected groups High). HPA rates the tissue IHC evidence Approved, with medium consistency between staining and RNA expression (HPA: reliability).
| Clear cytoplasmic stain in appendix or breast glandular cells, with cellular outlines still readable. | This fits the reported IHC pattern: both cell groups are High, within a broader cytoplasmic tissue profile (HPA: appendix and breast glandular cells High; cytoplasmic expression in all tissues). Judge it against controls on the same run; a single positive slide does not establish antibody specificity (standard IHC practice). |
| Fine cytoplasmic or perinuclear emphasis, without a distinct ciliary dot at routine IHC magnification. | Golgi and centrosomal association can contribute to local emphasis (UniProt Q8IY31 localization). A visible cilium is not required to call paraffin-section IHC positive: HPA describes tissue staining as cytoplasmic, while its resolved ciliary and microtubule locations come from ICC-IF (HPA: tissue IHC profile; subcellular ICC-IF). |
| Predominantly nuclear, crisp surface-membrane or extracellular staining with little cytoplasmic signal. | That distribution conflicts with the reported cytoplasmic IHC profile and the lack of a transmembrane segment (HPA: tissue IHC profile; UniProt Q8IY31 topology). Check morphology and controls before calling it target staining; chromogen deposits or nonspecific binding can mimic compartment-restricted signal (standard IHC practice). |
| Strong stain confined to an unexpected cell group while expected positive cells in the same section are blank. | Consider cross-reactivity or endogenous detection activity, especially if the discordance persists in controls (standard IHC practice). Broad expression alone cannot identify a cell group as truly negative: HPA reports cytoplasmic expression across tissues and provides no negative tissue group here (HPA: tissue IHC profile). |
| Uniform haze, or no signal in a known High group such as bronchial respiratory epithelium. | Haze obscures cell localization; absence in a reported High group makes a negative result hard to interpret (HPA: bronchus respiratory epithelial cells High). Assess the no-primary control, tissue integrity and run performance before assigning either result to IFT20 biology (standard IHC practice). |
💡Expected IFT20 appearanceCall a result consistent with IFT20 when cellular cytoplasm is distinctly stained in a reported High group, such as bronchial respiratory epithelium, while the no-primary control is clear; isolated nuclear, extracellular or uniform haze is suspect (HPA: bronchus High; cytoplasmic tissue profile; standard IHC practice).
| Tissue and cell-group selection | Appendix, breast, endometrium, epididymis and gallbladder glandular cells are High; bronchial respiratory epithelium is also High (HPA: tissue IHC levels). Cholangiocytes and the listed muscle cell groups are Low, so weak staining there is less decisive than a failure in a High group (HPA: tissue IHC levels). |
| Compartment and optical resolution | The tissue IHC summary is cytoplasmic; ICC-IF additionally resolves microtubules and primary cilia (HPA: tissue IHC profile; subcellular ICC-IF). Golgi, centrosome and basal-body association gives biological context, but paraffin IHC need not display each small structure separately (UniProt Q8IY31 localization; standard IHC practice). |
| Strength of validation | The listed antibody HPA021376 is Approved for IHC and ICC, while tissue staining has medium consistency with RNA expression (HPA: antibody status; reliability description). Treat a matching pattern as supportive evidence, and use controls to evaluate staining in the particular run (standard IHC practice). |
| Isoforms and epitope coverage | Four isoforms are listed, but this record gives no antibody epitope or isoform-specific IHC pattern (UniProt Q8IY31 isoforms; supplied HPA antibody record). Do not interpret regional intensity differences as isoform switching without separate evidence (standard IHC interpretation). |
| Topology and processing | IFT20 has no annotated transmembrane segment, signal peptide, propeptide or glycosylation site; its annotated chain spans residues 1–132 (UniProt Q8IY31 topology and processing). These facts support an intracellular interpretation but do not establish antigen-retrieval needs or fixation sensitivity (UniProt Q8IY31; supplied source scope). |
| Situation | Likely cause | Next action |
|---|---|---|
| A reported High tissue is completely blank. | A failed staining run, unsuitable section or detection problem is possible; HPA's High label does not guarantee every specimen or section will stain (HPA: selected High groups; standard IHC practice). | Inspect tissue preservation and the run's positive control; verify retrieval, primary-antibody application and chromogenic detection against the validated IHC procedure before interpreting absence (standard IHC practice). |
| Brown signal blankets tissue and obscures cell borders. | Background from detection chemistry, endogenous activity or insufficient washing can hide the expected cytoplasmic pattern (HPA: tissue IHC profile; standard IHC practice). | Compare no-primary and reagent controls; check the relevant endogenous-activity block, washing and detection development, then reassess identifiable cells (standard IHC practice). |
| Stain is mainly nuclear, extracellular or sharply membrane-limited. | The compartment disagrees with the cytoplasmic tissue pattern; IFT20 has no annotated transmembrane segment (HPA: tissue IHC profile; UniProt Q8IY31 topology). | Review section morphology and negative controls, and repeat with the IHC-validated antibody under its validated conditions if the mismatch persists (standard IHC practice). |
| A Low cell group appears stronger than nearby reported High cells. | Sample variation, nonspecific binding or endogenous detection activity may contribute; HPA labels cholangiocytes and listed muscle groups Low, not negative (HPA: tissue IHC levels; standard IHC practice). | Score cell groups separately, compare matched controls and avoid treating one intensity reversal as proof of altered IFT20 expression (HPA: tissue IHC levels; standard IHC practice). |
| Only scattered dots are visible, with no convincing cellular cytoplasm. | IFT20 associates with centrosomes and basal bodies, but isolated dots may also be deposits or background (UniProt Q8IY31 localization; standard IHC practice). | Check whether dots follow cell morphology, compare no-primary controls and seek a reproducible cytoplasmic pattern in a reported High group (HPA: tissue IHC profile; standard IHC practice). |
| Q: Should IF/ICC show the same broad chromogenic pattern? | A: IF/ICC offers finer compartment resolution; HPA reports approved microtubule and primary-cilium localization, with additional approved sites including basal body and mitotic spindle (HPA: subcellular ICC-IF). | Use the separate IF/ICC guide to interpret fluorescence localization; do not require individually resolved cilia in paraffin-section IHC (HPA: subcellular ICC-IF; tissue IHC profile; standard microscopy practice). |
Experiment evidence
IHC & IF images showing IFT20 staining
A07253 · IHCIHC analysis of IFT20 using anti-IFT20 antibody (A07253). IFT20 was detected in a paraffin-embedded section of human colon cancer tissue. He
A07253 · IHCIHC analysis of IFT20 using anti-IFT20 antibody (A07253). IFT20 was detected in a paraffin-embedded section of human colon cancer tissue. He
A07253 · IFIF analysis of IFT20 using anti-IFT20 antibody (A07253). IFT20 was detected in a paraffin-embedded section of human colon cancer tissue. Hea
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000109083-IFT20/tissue/cerebral+cortex#imid_5716836
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000109083-IFT20/tissue/colon#imid_5716762
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000109083-IFT20/tissue/liver#imid_5716763
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000109083-IFT20/tissue/kidney#imid_5716780
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA021376; green = IFT20, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA021376; green = IFT20, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for IFT20 IHC & IF
🧪Run bronchus first and score respiratory epithelial cells for IFT20 staining (HPA: High in bronchus respiratory epithelial cells). HPA detects IFT20 in all 45 scored tissues, so there is no supported negative tissue; use no-primary and isotype controls, and treat unstained cells within the bronchus section as local background rather than a validated biological negative (HPA: no negative rows).
Positive control tissue: Appendix (Glandular cells, HPA High)
Negative control tissue: None in HPA: IFT20 is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead.
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show IFT20 in A-431, U-251MG, U2OS, ASC52telo, RPTEC/TERT1, hTERT-RPE1 (serum starved), Sperm, KOLF2.1J, with annotated localisation: Microtubules (approved), Primary cilium (approved) (HPA subcellular).
Technical controls: Run secondary-only and host-matched rabbit IgG isotype controls alongside an IFT20-knockout specimen if available (caption: rabbit primary antibody; standard IHC control practice). Block endogenous peroxidase and inspect inflammatory cells for residual DAB signal in the bronchus section (standard chromogenic IHC practice).
⚠️Feasibility: No target-specific fixation window or fixation effect is reported, and the selected A07253 paraffin-section caption does not state a fixative (A07253 caption). The caption uses heat retrieval in EDTA at pH 8.0, but does not establish that retrieval is required (A07253 caption). Whether frozen sections or IF are easier is unreported; IF can help assess the expected ciliary localisation, while endogenous peroxidase may complicate chromogenic scoring in bronchus (HPA: primary cilium localisation; standard IHC practice).
HPA tissue IHC evidence for IFT20
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — 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 |
|---|---|---|---|---|
| Appendix | Glandular cells | High | Protein (IHC) | HPA → |
| Breast | Glandular cells | High | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | High | Protein (IHC) | HPA → |
| Cerebral cortex | Neuropil | High | Protein (IHC) | HPA → |
| Endometrium | Glandular cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| None in HPA: IFT20 is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead. | ||||
Section 3
Advanced IFT20 IHC Tips
Troubleshoot IFT20 staining in paraffin section IHC using the catalog antibody’s tested retrieval conditions and compartment aware controls.
Which retrieval conditions should I start with for weak IFT20 staining?
Start with heat mediated antigen retrieval in EDTA at pH 8.0 for paraffin sections (datasheet A07253). The documented image used 2 μg/ml primary antibody overnight at 4°C, so compare retrieval changes while holding antibody incubation constant (datasheet A07253). If staining remains weak, test a different retrieval buffer on matched serial sections as a controlled fallback, and record heating and cooling times (general IHC practice). Judge improvement by cellular staining with preserved morphology and matched negative controls; stronger staining alone can reflect increased background (general IHC practice).
How should I handle uncertain fixation when IFT20 staining is weak?
The A07253 paraffin section caption does not state a fixative, and target specific fixation sensitivity is unknown (datasheet A07253). Record each specimen’s fixative, fixation duration and processing history before comparing staining across blocks (general IHC practice). Use matched sections and keep EDTA retrieval at pH 8.0, the antibody incubation and chromogenic development constant while assessing suspected fixation effects (datasheet A07253; general IHC practice). If morphology or signal differs, report the association with processing rather than assigning a specific IFT20 fixation effect without a controlled comparison (general IHC practice).
Where should I expect IFT20 staining in paraffin sections?
Expect predominantly cytoplasmic tissue staining, with Golgi, centrosomal and ciliary pools possible where those structures can be resolved (HPA tissue IHC: cytoplasmic expression in all tissues; UniProt Q8IY31 subcellular). IFT20 is associated with the basal body at the base of primary cilia and with centrosomes during the cell cycle (UniProt Q8IY31 subcellular). Assess punctate or polarized staining against the cell outline and morphology before assigning it to either structure (general IHC practice). Diffuse chromogenic signal may establish cellular expression, but a small punctum in a paraffin section cannot by itself establish ciliary localization (general IHC practice).
Can this antibody distinguish IFT20 isoforms or epitope masking?
IFT20 has 4 annotated isoforms, but the supplied A07253 caption does not identify the antibody epitope or its isoform coverage (UniProt Q8IY31 isoforms; datasheet A07253). Do not interpret a missing chromogenic signal as absence of every isoform without an epitope map and independent validation (general IHC practice). The annotated protein lacks a transmembrane segment and reported glycosylation sites, yet these annotations do not establish how its epitope survives tissue processing (UniProt Q8IY31 topology and glycosylation). Compare serial sections under controlled retrieval and antibody conditions, and describe the result as antibody detected IFT20 staining rather than isoform specific expression (general IHC practice).
How can I check a putative ciliary IFT20 signal by multiplex IF?
Use a marker of the expected cell type alongside a ciliary or basal body marker to place IFT20 signal within the correct cell and structure (UniProt Q8IY31 subcellular; HPA subcellular). Choose spectrally separated fluorophores and inspect an unstained tissue section so autofluorescence is not mistaken for a small punctum (general IF practice). Because IFT20 has no annotated transmembrane segment and includes cytoplasmic and ciliary pools, permeabilize to access intracellular epitopes, then optimize its strength against morphology and background (UniProt Q8IY31 topology and subcellular; general IF practice). Treat IF colocalization as supporting spatial evidence rather than proof that a chromogenic IHC punctum marks a cilium (general IF and IHC practice).
What should I change when IFT20 DAB staining is diffuse?
First compare the tissue with a no primary control and inspect whether signal tracks edges, damaged areas or endogenous pigment (general IHC practice). The documented A07253 section used 10% goat serum blocking and a peroxidase linked secondary followed by DAB detection (datasheet A07253). Include an appropriate endogenous peroxidase block, then titrate antibody concentration and DAB development on matched sections while keeping retrieval fixed at EDTA pH 8.0 (general IHC practice; datasheet A07253). Because broad cytoplasmic IFT20 expression is reported, diffuse cellular signal requires morphology and controls before it is classified as nonspecific background (HPA tissue IHC: cytoplasmic expression in all tissues; general IHC practice).
How should I score IFT20 across paraffin section samples? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and analysis region before scoring, and exclude folds, necrosis and poorly preserved tissue by a rule applied across samples (general IHC practice). For diffuse cellular staining, record percent positive cells and an H score from 0 to 300 using intensity grades 0 to 3 (general IHC practice). If discrete puncta are reliably resolvable, report their density per mm² and verify that the same detection settings were used throughout (general IHC practice). Normalize cellular scores to evaluable cells and punctum counts to analyzed tissue area or the relevant cell count, then report compartments separately (general IHC practice; UniProt Q8IY31 subcellular).
How can I distinguish genuine IFT20 staining from artefact?
Check whether staining falls within identifiable cells and plausible cytoplasmic, Golgi associated or basal body regions, while allowing for compartment resolution limits in paraffin sections (HPA tissue IHC; UniProt Q8IY31 subcellular; general IHC practice). Strong signal confined to tissue edges, necrotic areas or no primary controls should prompt investigation of processing or detection artefact (general IHC practice). Endogenous peroxidase can produce misleading DAB signal unless appropriately blocked and controlled (general IHC practice). Broad cytoplasmic expression is reported, and HPA rates tissue staining as approved with medium RNA agreement, so use controls and morphology before calling a case IFT20 negative or a punctum ciliary (HPA tissue IHC; general IHC practice).
Boster reagents
Best IFT20 / Intraflagellar transport protein 20 homolog IHC Antibodies
A07253 has IHC and IF images from paraffin-embedded human colon cancer tissue, plus an IHC image from human testis cancer tissue (catalog image captions).
Real IHC data
Anti-IFT20 Antibody ®
Cat # A07253
A07253 is listed for IHC and IF and for human, mouse, and rat reactivity (catalog: applications and reactivity). Its IHC captions show paraffin-embedded human colon and testis cancer sections; its IF caption shows a paraffin-embedded human colon cancer section (catalog image captions).
Which to pick: Choose A07253 for paraffin-section IHC: its colon cancer caption documents EDTA retrieval at pH 8.0 and 2 μg/ml primary antibody (A07253 IHC caption). For IF, A07253 has a paraffin-section example at 5 μg/ml; ICC validation and clonality are unreported (A07253 IF caption; catalog: applications and clone). For mouse or rat work, A07253 lists both species as reactive, while its IHC images show human tissue only; the fixative is unreported (catalog: reactivity; A07253 IHC captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q8IY31 (IFT20_HUMAN, Intraflagellar transport protein 20 homolog).
- Human Protein Atlas. IFT20 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. IFT20 subcellular location (ICC-IF): Mainly localized to the microtubules and primary cilium. In addition localized to the mitotic spindle, basal body, flagellar centriole, mid piece, principal piece and end piece..
- Human Protein Atlas. IFT20 antibody validation summary (1 antibodies).
- IFT20 is critical for collagen biosynthesis in craniofacial bone formation. Biochemical and biophysical research communications 2020 — PMC7744399.
- IFT20 governs mesenchymal stem cell fate through positively regulating TGF-β-Smad2/3-Glut1 signaling mediated glucose metabolism. Redox biology 2022 — PMC9243161.
- IFT20 is required for the maintenance of cartilaginous matrix in condylar cartilage. Biochemical and biophysical research communications 2019 — PMC6443088.
- Associations of IFT20 and GM130 protein expressions with clinicopathological features and survival of patients with lung adenocarcinoma. BMC cancer 2022 — PMC9308367.
- PubMed PMID:14672413 — UniProt-cited evidence.
- PubMed PMID:12107411 — UniProt-cited evidence.
- PubMed PMID:16625196 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for IFT20 (Intraflagellar transport protein 20 homolog). Expected localisation Cytoplasmic tissue staining (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A07253). Validated IHC antibody: A07253 at 2-5 μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Appendix (Glandular cells); negative None in HPA (detected in all 45 tissues); use no-primary + isotype controls. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human colon cancer tissue; fixative not specified (datasheet A07253); 2. Fixation: Image fixative and duration unreported (datasheet A07253); 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 A07253); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A07253); 8. Primary antibody: Rabbit anti-IFT20, 2-5 μg/ml (datasheet A07253); 9. Primary incubation: Overnight at 4 °C (datasheet A07253). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific); Caveat: Staining and RNA show medium consistency (HPA tissue IHC); Isoform / epitope: Four isoforms; epitope coverage is unreported (UniProt); Regulation: Low tissue specificity (HPA tissue RNA). Evidence: UniProt Q8IY31, HPA tissue IHC · Approved, HPA ICC-IF · Mainly localized to the microtubules and primary cilium. In addition…, Datasheet A07253, PMC9308367. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/IFT20/immunohistochemistry-ihc-guide.