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IFFO1 / Non-homologous end joining factor IFFO1 · IHC design guide
Design Immunohistochemistry for IFFO1
Plan IFFO1 chromogenic IHC using the catalog antibody at 2–5 μg/ml (datasheet A17273-1). Compare staining with the reported nuclear and cytoplasmic tissue pattern, while accounting for its low consistency with RNA expression (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
IFFO1 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 and cytoplasmic staining observed (HPA tissue IHC) | |
| Staining pattern | Several tissues: nuclear and cytoplasmic staining (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A17273-1) | |
| Positive control | Testis+4 more · see all | |
| Negative control | Appendix+4 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep paraffin-section fixation consistent. (standard IHC practice; not target-specific) | |
| Caveat | Staining has low consistency with RNA data (HPA tissue IHC) | |
| Regulation | Ubiquitously expressed (UniProt) | |
| Isoform / epitope | 7 isoforms; check epitope coverage (UniProt) |
Section 1
Recommended IFFO1 IHC & IF Protocols
Compare the catalog antibody’s IHC-P protocol (datasheet A17273-1) with one published IFFO1 paraffin-section protocol (PMC12241364).
| Sample | Paraffin-embedded human esophageal squamous carcinoma tissue; fixative not specified (datasheet A17273-1) |
| Fixation | Image fixative and duration unreported (datasheet A17273-1); 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 A17273-1); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A17273-1) |
| Primary antibody | Rabbit anti-IFFO1, 2-5 μg/ml (datasheet A17273-1) |
| Primary incubation | Overnight at 4 °C (datasheet A17273-1) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet A17273-1) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | IFFO1-positive staining in pachytene spermatocytes of testis (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic and nuclear expression in several tissues. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA retrieval at pH 8.0 (datasheet A17273-1); the published protocol does not specify retrieval (PMC12241364).
Section 2
What Is the Expected IFFO1 Staining Pattern?
IFFO1 is chiefly nuclear, with nucleoplasmic, inner nuclear membrane and nuclear matrix localization; it has no transmembrane segment (UniProt Q0D2I5). In IHC, expect nuclear and some cytoplasmic staining across several tissues, strongest in testis pachytene spermatocytes (HPA tissue IHC: High). Treat this as a provisional pattern: HPA rates the antibody Approved but reports low consistency with RNA data and pending external verification (HPA tissue IHC: reliability).
| Strong nuclear staining in testis pachytene spermatocytes, with possible cytoplasmic staining (HPA tissue IHC: High; cytoplasmic and nuclear profile). | This best matches the supplied positive reference. Nuclear signal fits IFFO1 localization (UniProt Q0D2I5 subcellular location); cytoplasmic signal also occurs in HPA tissue IHC, so assess compartment, cell type and controls together before assigning specificity (HPA tissue IHC: profile; general IHC practice). |
| Moderate staining in cerebral cortex neurons, colon or duodenum glandular cells, or kidney tubule cells (HPA tissue IHC: Medium). | These are additional observed positive settings, useful when testis is unavailable (HPA tissue IHC: positive cells). Match the named cell population and compare with a negative control; intensity alone cannot resolve specificity because the HPA antibody is Approved with low RNA–staining consistency (HPA tissue IHC: reliability; general IHC practice). |
| Predominantly extracellular or plasma membrane staining, with little nuclear signal (UniProt Q0D2I5 subcellular location and topology). | This is discordant with the reported nuclear localization and lack of a transmembrane segment; consider an artefact or off-target signal (UniProt Q0D2I5 subcellular location and topology; general IHC interpretation). A nuclear rim could instead reflect the reported inner nuclear membrane location (UniProt Q0D2I5 subcellular location). |
| Strong signal in appendix glandular cells or bone marrow hematopoietic cells (HPA tissue IHC: Not detected). | Those populations were not detected in the supplied HPA observations; strong staining calls for review of cell identification, background, cross-reactivity and endogenous detection activity (HPA tissue IHC: negative cells; general IHC practice). A negative HPA observation does not prove that every specimen must be negative (HPA tissue IHC: reliability). |
| Uniform haze over cells and tissue, obscuring nuclei and cell boundaries (general IHC practice). | A diffuse field cannot be scored reliably as IFFO1 localization; assess nonspecific binding, incomplete washing and chromogenic detection background with appropriate controls (general IHC practice). The HPA pattern is cell-associated nuclear and cytoplasmic staining, rather than a uniform field (HPA tissue IHC: profile). |
💡Expected IFFO1 appearanceA convincing positive is strong, cell-associated nuclear staining in testis pachytene spermatocytes, with possible cytoplasmic signal (HPA tissue IHC: High; cytoplasmic and nuclear profile); widespread extracellular or plasma membrane-only color is suspect (UniProt Q0D2I5 subcellular location and topology; general IHC interpretation).
| Compartment and topology (UniProt Q0D2I5 subcellular location and topology) | IFFO1 is mainly soluble nuclear protein, with a remaining nuclear matrix fraction and an inner nuclear membrane location; it has no transmembrane segment (UniProt Q0D2I5). Interpret a nuclear rim in that context, while using the observed HPA cytoplasmic staining as a qualified tissue finding (HPA tissue IHC: profile). |
| Antibody evidence (HPA antibodies: HPA069344 IHC Approved; HPA tissue IHC: reliability) | The listed antibody is Approved for IHC, yet HPA reports low agreement between staining and RNA expression and pending external verification (HPA antibodies; HPA tissue IHC: reliability). Use the cell-specific positive and negative observations as comparison points, not proof that every stained cell contains IFFO1 (HPA tissue IHC: positive and negative cells). |
| Alternative isoforms (UniProt Q0D2I5 isoforms 1–7) | Seven isoforms are listed, but the supplied record gives no antibody epitope or isoform coverage (UniProt Q0D2I5 isoforms; supplied antibody record). Do not infer that this antibody detects every isoform or assign a tissue intensity difference to a particular isoform (general IHC interpretation). |
| Antigen retrieval and fixation sensitivity (general IHC practice) | Retrieval is a routine variable in paraffin IHC, but no IFFO1-specific retrieval condition or fixation effect is supplied (general IHC practice; supplied evidence). Optimize conditions against known positive tissue and controls without treating a change in staining as evidence of a target-specific fixation mechanism (HPA tissue IHC: testis High; general IHC practice). |
| IF/ICC Q&A: Is an expected cell-line pattern established? (HPA subcellular ICC-IF) | No: HPA lists no main ICC-IF location and no cell lines with ICC-IF images (HPA subcellular ICC-IF). UniProt supports a nuclear localization hypothesis, but the supplied ICC-IF record cannot validate an IF image or justify an IF protocol option (UniProt Q0D2I5 subcellular location; HPA subcellular ICC-IF). |
| Situation | Likely cause | Next action |
|---|---|---|
| No signal in testis pachytene spermatocytes (HPA tissue IHC: High). | The expected positive population may be absent from the section, or the IHC run may lack sensitivity (HPA tissue IHC: positive cells; general IHC practice). | Confirm the cells are present, review reagent and detection controls, then assess retrieval and antibody concentration within a controlled IHC-P optimization (general IHC practice); no target-specific setting is supplied (supplied evidence). |
| Only cytoplasmic staining, with nuclei consistently blank (HPA tissue IHC: profile; UniProt Q0D2I5 location). | The result is incomplete relative to UniProt nuclear localization, although HPA does report cytoplasmic tissue staining (UniProt Q0D2I5 subcellular location; HPA tissue IHC: profile). | Compare with a testis positive section and detection controls; review retrieval and scoring before attributing the cytoplasmic signal to IFFO1 (HPA tissue IHC: testis High; general IHC practice). |
| Strong staining in appendix glandular cells (HPA tissue IHC: Not detected). | Cell identification, nonspecific binding or endogenous chromogenic detection activity may account for an unexpected positive (HPA tissue IHC: negative cells; general IHC practice). | Check morphology and controls, including a primary-antibody omission control; compare the same run with testis pachytene spermatocytes (general IHC practice; HPA tissue IHC: testis High). |
| Diffuse brown background masks cell boundaries (general IHC practice). | Nonspecific reagent binding, insufficient washing or detection background may obscure the cell-associated HPA pattern (general IHC practice; HPA tissue IHC: profile). | Review blocking, washing and detection controls, then score only interpretable cells with visible boundaries and a counterstain (general IHC practice). |
| A medium-positive tissue stains less strongly than testis (HPA tissue IHC: Medium versus High). | This can agree with the supplied HPA categories; for example, colon glandular cells are Medium while testis pachytene spermatocytes are High (HPA tissue IHC: positive cells). | Score the specified cell populations separately and compare slides processed in the same run; avoid converting HPA categories into quantitative expression measurements (general IHC practice; HPA tissue IHC: reliability). |
| An IF/ICC image appears positive but its compartment is unclear (HPA subcellular ICC-IF: no images). | The supplied HPA ICC-IF record provides neither a validated main location nor example cell-line images (HPA subcellular ICC-IF). | Use the separate IF/ICC guide for method choices; treat nuclear localization as a UniProt-based expectation and seek independent image controls before claiming IF validation (UniProt Q0D2I5 subcellular location; HPA subcellular ICC-IF; general IF practice). |
Experiment evidence
IHC & IF images showing IFFO1 staining
A17273-1 · IHCIHC analysis of IFFO1 using anti-IFFO1 antibody (A17273-1). IFFO1 was detected in a paraffin-embedded section of human esophageal squamous c
A17273-1 · IHCIHC analysis of IFFO1 using anti-IFFO1 antibody (A17273-1). IFFO1 was detected in a paraffin-embedded section of human rectum adenocarcinoma
A17273-1 · IFIF analysis of IFFO1 using anti-IFFO1 antibody (A17273-1) and anti-Beta Tubulin antibody (M01857-3). IFFO1 was detected in immunocytochemica
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000010295-IFFO1/tissue/cerebral+cortex#imid_19170263
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000010295-IFFO1/tissue/colon#imid_19170189
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000010295-IFFO1/tissue/liver#imid_19170190
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000010295-IFFO1/tissue/kidney#imid_19170199
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for IFFO1 IHC & IF
🧪Run testis first and look for staining in pachytene spermatocytes (HPA: High in pachytene spermatocytes). Use appendix glandular cells as a negative tissue (HPA: Not detected); on the testis slide, assess morphologically identified non-pachytene cells for low or absent staining without assuming every other cell type is negative.
Positive control tissue: Testis (Pachytene spermatocytes, HPA High)
Negative control tissue: Appendix (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA carries no ICC-IF cell line for IFFO1; derive a cell-line control from the positive tissue's cell type (Pachytene spermatocytes) and confirm it by RNA or western blot first.
Technical controls: Include a no-primary, secondary-only control, a concentration-matched rabbit IgG isotype control for the rabbit primary (selected tissue-IHC caption), and IFFO1 knockout tissue as a biological negative if available. Check endogenous peroxidase background on the testis section before interpreting DAB staining (standard chromogenic IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected A17273-1 paraffin-section caption does not state a fixative. Heat retrieval in EDTA at pH 8.0 was used in that caption, but a requirement for retrieval has not been established. The supplied evidence does not establish whether frozen sections or IF/ICC are easier, or identify a testis-specific artefact; HPA lists no ICC-IF images for IFFO1 (HPA subcellular).
HPA tissue IHC evidence for IFFO1
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — 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 | Pachytene spermatocytes | High | Protein (IHC) | HPA → |
| Cerebral cortex | Neuronal cells | Medium | Protein (IHC) | HPA → |
| Colon | Glandular cells | Medium | Protein (IHC) | HPA → |
| Duodenum | Glandular cells | Medium | Protein (IHC) | HPA → |
| Kidney | Cells in tubules | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Appendix | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Cervix | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Endometrium | Cells in endometrial stroma | Not detected | Protein (IHC) | HPA → |
| Epididymis | Glandular cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced IFFO1 IHC Tips
Use compartment aware controls to interpret IFFO1 staining in paraffin sections; the catalog antibody has a documented chromogenic IHC example (datasheet A17273-1).
What retrieval should I try first if nuclear IFFO1 staining is weak?
Start with heat mediated antigen retrieval in EDTA at pH 8.0 for paraffin sections (datasheet A17273-1). The documented example used this retrieval before overnight incubation at 4°C with 2 μg/ml catalog antibody, so use that combination as the initial comparison point (datasheet A17273-1). If staining remains weak, compare a shorter and a longer heating interval on matched sections while keeping the antibody and detection conditions fixed (general IHC practice). Judge the result by nuclear signal in intact cells and by preservation of tissue morphology, since IFFO1 is reported in the nucleoplasm and nuclear matrix (UniProt Q0D2I5).
Could fixation explain inconsistent IFFO1 staining between paraffin blocks?
The selected paraffin section example does not state its fixative, so target specific fixation sensitivity for IFFO1 is unknown (datasheet A17273-1). Record each block’s fixative and fixation duration when available, then compare sections with similar processing and the same EDTA pH 8.0 retrieval (general IHC practice; datasheet A17273-1). Include a processed positive control in each run to separate a block related loss of signal from a staining run failure (general IHC practice). Do not infer fixation tolerance from reported tissue staining or from the protein’s lack of a transmembrane segment (HPA tissue IHC; UniProt Q0D2I5 topology).
Should I score cytoplasmic staining as IFFO1 positive?
Prioritize nuclear staining, including nucleoplasmic or nuclear matrix patterns, when assessing the expected IFFO1 compartment (UniProt Q0D2I5 subcellular location). IFFO1 is reported at double strand break sites near lamina and nuclear matrix structures, but routine tissue IHC cannot establish that a stained nuclear focus marks a repair site (UniProt Q0D2I5; general IHC practice). HPA reports both cytoplasmic and nuclear tissue staining, with low consistency between antibody staining and RNA expression data (HPA tissue IHC). Score cytoplasmic signal separately, and require an independent specificity control before treating cytoplasmic only staining as target specific (general IHC practice).
Can this stain distinguish IFFO1 isoforms or reveal epitope loss?
IFFO1 has 7 listed isoforms and an intermediate filament rod spanning residues 73–526 in the supplied record (UniProt Q0D2I5). The selected caption provides staining conditions but no epitope location or isoform reactivity, so this IHC result cannot assign a stained cell to a particular isoform (datasheet A17273-1). If signal differs between blocks, first compare retrieval and processing controls before attributing the difference to an epitope change (general IHC practice). An epitope mapped by the antibody documentation, together with an isoform aware independent assay, would be needed to test that interpretation (general IHC practice).
How should I use IF to check a surprising chromogenic IFFO1 pattern?
Use IF as a separate follow up to the paraffin section IHC result, since the supplied HPA subcellular record lists no ICC/IF images for IFFO1 (HPA subcellular). Multiplex IFFO1 with a marker of the cell population being evaluated and a nuclear counterstain, then compare signal within identified cells (general IF practice). Choose a fluorophore whose emission can be separated from the tissue’s autofluorescence, and include single stain controls when assessing overlap (general IF practice). For an intracellular nuclear epitope, include a controlled permeabilisation step; IFFO1 has no reported transmembrane segment, while antibody epitope accessibility remains unspecified (UniProt Q0D2I5 topology; general IF practice).
How can I reduce diffuse DAB or edge staining without losing nuclear signal?
The documented IHC example used goat serum at 10%, a peroxidase linked secondary antibody, and DAB development (datasheet A17273-1). Add an endogenous peroxidase block and a no primary control to assess enzyme driven colour and nonspecific detection in the same tissue batch (general chromogenic IHC practice). If diffuse staining persists, titrate the primary around the documented 2 μg/ml condition and compare shorter chromogen development on matched sections (datasheet A17273-1; general IHC practice). Treat staining concentrated at cut edges or damaged areas cautiously, and retain settings that preserve interpretable nuclear signal in intact cells (general IHC practice; UniProt Q0D2I5 subcellular location).
What should I measure when comparing IFFO1 staining across specimens? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and nuclear scoring rule before comparing slides, because IFFO1 has a reported nuclear location while HPA also describes cytoplasmic tissue staining (UniProt Q0D2I5; HPA tissue IHC). Record the percentage of positive nuclei and an intensity based H-score, or report positive nuclear density per mm² when cell counts differ (general IHC practice). Normalize the first measures to evaluable cells of the same population, or normalize density to analyzed viable tissue area (general IHC practice). Keep cytoplasmic scores separate and use matched retrieval, detection, and image acquisition settings across the comparison (general IHC practice).
What evidence would make an IFFO1 positive result convincing?
A convincing result shows reproducible staining in intact nuclei under the documented EDTA pH 8.0 retrieval conditions, with an appropriate no primary control (datasheet A17273-1; UniProt Q0D2I5 subcellular location; general IHC practice). HPA reports high staining in testis pachytene spermatocytes, but flags low consistency between antibody staining and RNA expression data, so use its pattern as context rather than proof (HPA tissue IHC). Investigate cytoplasmic only signal, unexpected cell populations, section edges, and necrotic areas with matched controls (general IHC practice). Exclude endogenous peroxidase colour before interpreting DAB signal as IFFO1, and seek an independent specificity check for unexpected patterns (general chromogenic IHC practice).
Boster reagents
Best IFFO1 / Non-homologous end joining factor IFFO1 IHC Antibodies
A17273-1 has IHC data from human paraffin sections and IF/ICC data from A549 cells; its listed reactivity is human, mouse and rat (catalog; image captions).
Real IHC data
Anti-IFFO1 Antibody ®
Cat # A17273-1
A17273-1 was shown in IHC on paraffin sections of human esophageal squamous carcinoma and rectum adenocarcinoma (IHC image captions). A17273-1 was shown in IF/ICC on A549 cells (IF image caption); the catalog lists human, mouse and rat reactivity.
Which to pick: Choose A17273-1 for paraffin-section IHC using 2–5 μg/ml as the catalog range; its IHC captions show human tissue sections with EDTA pH 8.0 retrieval (catalog; IHC image captions). A17273-1 also supports IF/ICC at 5 μg/ml in the catalog, with an A549 IF image; mouse and rat are listed as reactive species, but the supplied IHC and IF images show human samples only (catalog; image captions). Clonality and the tissue fixative are unreported (catalog; IHC image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q0D2I5 (IFFO1_HUMAN, Non-homologous end joining factor IFFO1).
- Human Protein Atlas. IFFO1 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. IFFO1 subcellular location (ICC-IF): Highest expression in LHCN-M2: 16.7 nTPM.
- Human Protein Atlas. IFFO1 antibody validation summary (1 antibodies).
- Intermediate filament IFFO1 negatively regulates the migration of lung cancer cells by inhibiting the IQGAP3-Cdc42 interaction. Cell death & disease 2025 — PMC12241364.
- PubMed PMID:15489334 — UniProt-cited evidence.
- PubMed PMID:12032826 — UniProt-cited evidence.
- PubMed PMID:31548606 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for IFFO1 (Non-homologous end joining factor IFFO1). Expected localisation Nuclear and cytoplasmic staining observed (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A17273-1). Validated IHC antibody: A17273-1 at 2-5 μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Testis (Pachytene spermatocytes); negative Appendix. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human esophageal squamous carcinoma tissue; fixative not specified (datasheet A17273-1); 2. Fixation: Image fixative and duration unreported (datasheet A17273-1); 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 A17273-1); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A17273-1); 8. Primary antibody: Rabbit anti-IFFO1, 2-5 μg/ml (datasheet A17273-1); 9. Primary incubation: Overnight at 4 °C (datasheet A17273-1). Watch for: Fixation: Keep paraffin-section fixation consistent. (standard IHC practice; not target-specific); Caveat: Staining has low consistency with RNA data (HPA tissue IHC); Isoform / epitope: 7 isoforms; check epitope coverage (UniProt); Regulation: Ubiquitously expressed (UniProt). Evidence: UniProt Q0D2I5, HPA tissue IHC · Approved, HPA cell-line RNA · Highest expression in LHCN-M2: 16.7 nTPM, Datasheet A17273-1, PMC12241364. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/IFFO1/immunohistochemistry-ihc-guide.