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SFXN3 / Sideroflexin-3 · IHC design guide
Design Immunohistochemistry for SFXN3
Plan chromogenic paraffin section IHC for SFXN3 using the catalog antibody at 2–5 μg/ml (datasheet A14648-1). Use high staining in hepatocytes as a positive reference (HPA tissue IHC), and interpret cytoplasmic and nuclear tissue staining in light of the mitochondrial membrane annotation (HPA tissue IHC; UniProt).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
SFXN3 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 | Tissue IHC shows cytoplasmic and nuclear staining; the protein is annotated at the mitochondrial membrane (HPA tissue IHC; UniProt) | |
| Staining pattern | High staining in hepatocytes; cytoplasmic and nuclear overall (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A14648-1) | |
| Positive control | Hippocampus+4 more · see all | |
| Negative control | Adipose tissue+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 | Nuclear IHC staining differs from the mitochondrial membrane annotation (HPA tissue IHC; UniProt) | |
| Regulation | Expression regulation is not annotated (UniProt) | |
| Isoform / epitope | No isoforms annotated; epitope orientation is unreported (UniProt) |
Section 1
Recommended SFXN3 IHC & IF Protocols
The catalog antibody’s IHC-P protocol is accompanied by a published SFXN3 thyroid-section protocol (PMC10290451).
| Sample | Paraffin-embedded human liver cancer tissue; fixative not specified (datasheet A14648-1) |
| Fixation | Image fixative and duration unreported (datasheet A14648-1); 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 A14648-1); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A14648-1) |
| Primary antibody | Rabbit anti-SFXN3, 2-5 μg/ml (datasheet A14648-1) |
| Primary incubation | Overnight at 4 °C (datasheet A14648-1) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet A14648-1) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | SFXN3-positive staining in neuronal cells of hippocampus (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic and nuclear expression in most tissues. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA pH 8.0 retrieval (datasheet A14648-1). The published protocol specifies 1 mM EDTA but gives no pH (PMC10290451).
Section 2
What Is the Expected SFXN3 Staining Pattern?
SFXN3 is a mitochondrial membrane protein with 4 transmembrane segments (UniProt Q9BWM7 topology). In paraffin IHC, assess cytoplasmic staining in cells reported as high, including kidney tubular cells, hepatocytes and lung macrophages (HPA: High). HPA also reports nuclear staining across most tissues; that observation needs cautious interpretation alongside mitochondrial localisation (HPA: tissue profile; HPA: ICC-IF mitochondria). Tissue IHC reliability is Approved, with medium consistency against RNA data (HPA: tissue IHC).
| Cytoplasmic, possibly granular staining in kidney tubular cells, hepatocytes or lung macrophages (HPA: High in each). | This fits the reported high IHC signal and the predicted mitochondrial compartment (HPA: tissue IHC; UniProt Q9BWM7 localisation). Granularity is an expectation from mitochondrial localisation, not a separately established HPA tissue IHC pattern. |
| Predominantly nuclear staining with little cytoplasmic signal (HPA: nuclear expression reported in most tissues). | HPA tissue IHC includes nuclear staining, while UniProt and HPA ICC-IF place SFXN3 in mitochondria (HPA: tissue profile; UniProt Q9BWM7; HPA: ICC-IF). Treat nuclear-only staining as unresolved; compare controls before assigning it to SFXN3. |
| Strong staining in adipocytes or marrow hematopoietic cells (HPA: Not detected in those cells). | This disagrees with the listed tissue IHC references and raises possible cross-reactivity or detection artefact (HPA: tissue IHC; general IHC practice). HPA reports low tissue specificity, so an unexpected cell alone does not establish a false positive (HPA: RNA specificity). |
| Diffuse color across cells and tissue spaces, obscuring cell boundaries (general IHC practice). | This is background rather than an interpretable cell pattern (general IHC practice). Blocking, washing or detection chemistry may contribute (general IHC practice); the supplied HPA and UniProt records do not identify a SFXN3-specific cause. |
| No signal in kidney tubular cells, hepatocytes or lung macrophages (HPA: High in each). | The result conflicts with HPA reference staining but does not prove that the tissue lacks SFXN3 (HPA: tissue IHC). Check the IHC-validated antibody, positive control and detection run before scoring the sample negative (HPA: HPA008028 IHC Approved; general IHC practice). |
💡Expected SFXN3 appearanceCall a result positive when identifiable high-reference cells show clear cytoplasmic staining compatible with mitochondrial localisation; treat dominant nuclear-only signal or equally strong staining in HPA not-detected cells as needing control-based review (HPA: tissue IHC and ICC-IF; UniProt Q9BWM7).
| Compartment reference | SFXN3 is assigned to the mitochondrial membrane, and HPA ICC-IF reports enhanced mitochondrial localisation (UniProt Q9BWM7; HPA: ICC-IF). Cytoplasmic IHC signal should be judged in that context; paraffin IHC need not resolve individual mitochondria (general IHC practice). |
| Membrane topology | The 4 annotated transmembrane segments support a membrane-associated target (UniProt Q9BWM7 topology). Their positions do not identify this antibody's epitope or establish a particular antigen retrieval condition (UniProt Q9BWM7 topology; general IHC practice). |
| Tissue and cell reference | HPA reports high staining in several specific cell populations, low staining in others, and no detection in selected cells (HPA: tissue IHC). Compare the same cell types when scoring; the reference levels are not guaranteed intensities for every IHC run (general IHC practice). |
| Evidence strength by application | HPA008028 is IHC Approved; HPA008028 and HPA048105 are ICC Enhanced (HPA: antibody validation). ICC validation supports the mitochondrial localisation assessment but does not upgrade tissue IHC beyond its Approved, medium-consistency status (HPA: antibody validation; HPA: tissue IHC). |
| Situation | Likely cause | Next action |
|---|---|---|
| High-reference cells show no detectable stain (HPA: High in kidney tubules, hepatocytes and lung macrophages). | The run may have a reagent, retrieval or detection problem (general IHC practice); HPA intensity alone does not identify which step failed (HPA: tissue IHC). | Inspect a positive tissue control and reagent controls, then review retrieval and detection against the IHC-validated antibody's instructions (general IHC practice; HPA: HPA008028 IHC Approved). |
| Nuclear signal dominates the slide (HPA: nuclear expression in most tissues). | HPA tissue IHC and mitochondrial localisation evidence differ on this compartment (HPA: tissue profile; HPA: ICC-IF; UniProt Q9BWM7). | Score cytoplasmic and nuclear compartments separately; review matched controls before calling nuclear-only signal specific (general IHC practice). |
| Adipocytes or marrow hematopoietic cells stain strongly (HPA: Not detected in those cells). | Cross-reactivity or detection activity is possible (general IHC practice), though HPA's reference pattern cannot diagnose the cause in a new specimen (HPA: tissue IHC). | Compare a high-reference tissue and a no-primary control; interpret the unexpected cells only after assessing background (HPA: tissue IHC; general IHC practice). |
| Color is diffuse or tissue-wide (general IHC practice). | Background may arise from blocking, washing or the detection system (general IHC practice); no SFXN3-specific background mechanism is supplied. | Review wash and blocking steps and a no-primary control; for enzyme detection, assess endogenous activity according to the detection chemistry (general IHC practice). |
| Only faint staining appears in a high-reference cell population (HPA: High in kidney tubules, hepatocytes and lung macrophages). | A weak run or differences from the HPA reference may explain the result (general IHC practice; HPA: tissue IHC). No target-specific fixation sensitivity is established here. | Check tissue integrity, positive control performance and the antibody's IHC instructions before interpreting faint signal as low expression (general IHC practice). |
| Can IF/ICC clarify a disputed compartment? | HPA ICC-IF reports enhanced mitochondrial localisation, whereas its tissue IHC profile includes cytoplasmic and nuclear staining (HPA: ICC-IF; HPA: tissue profile). | Use the separate IF/ICC guide for that application; compare its localisation evidence with the IHC result without treating it as an IHC protocol (HPA: ICC-IF; general IHC practice). |
Experiment evidence
IHC & IF images showing SFXN3 staining
A14648-1 · IHCIHC analysis of SFXN3 using anti-SFXN3 antibody (A14648-1). SFXN3 was detected in a paraffin-embedded section of human liver cancer tissue.
A14648-1 · IHCIHC analysis of SFXN3 using anti-SFXN3 antibody (A14648-1). SFXN3 was detected in a paraffin-embedded section of human lung adenocarcinoma t
A14648-1 · IFIF analysis of SFXN3 using anti-SFXN3 antibody (A14648-1). SFXN3 was detected in a paraffin-embedded section of human renal cancer tissue. H
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000107819-SFXN3/tissue/cerebral+cortex#imid_2444712
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000107819-SFXN3/tissue/colon#imid_2444654
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000107819-SFXN3/tissue/liver#imid_2444639
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000107819-SFXN3/tissue/kidney#imid_2444640
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA048105; green = SFXN3, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA048105; green = SFXN3, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for SFXN3 IHC & IF
🧪Run liver first: hepatocytes should stain (HPA: High in hepatocytes). Run adipose tissue as the negative comparator (HPA: Not detected in adipocytes); on the liver slide, non-hepatocyte cells without chromogen above background can serve as provisional internal references, but their SFXN3-negative status is unverified.
Positive control tissue: Hippocampus (Neuronal cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show SFXN3 in A-431, HUVEC/TERT2, U2OS, U-251MG, U2OS, siRNA 2 (10x), U2OS, siRNA 2 (40x), U2OS, scrambled (10x), U2OS, scrambled (40x), NIH 3T3, with annotated localisation: Mitochondria (enhanced) (HPA subcellular).
Technical controls: Include no-primary (secondary-only) and species-matched rabbit IgG isotype controls, plus SFXN3-knockout material as a biological negative (caption: rabbit primary; standard IHC practice). Quench endogenous peroxidase and check background in the liver section (standard chromogenic IHC practice).
⚠️Feasibility: A target-specific fixation window and fixation effect are unreported, and the selected A14648-1 caption does not state the fixative (selected-SKU caption). The reported paraffin-section staining used heat retrieval in EDTA at pH 8.0, followed by 2 μg/ml primary antibody overnight at 4°C; whether retrieval is strictly required is unreported (selected-SKU caption). Neither frozen sections nor tissue IF are established as easier by the supplied evidence; hepatocyte pigment and endogenous peroxidase can complicate chromogenic interpretation (standard IHC practice).
HPA tissue IHC evidence for SFXN3
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 |
|---|---|---|---|---|
| Hippocampus | Neuronal cells | High | Protein (IHC) | HPA → |
| Kidney | Cells in tubules | High | Protein (IHC) | HPA → |
| Liver | Hepatocytes | High | Protein (IHC) | HPA → |
| Lung | Macrophages | High | Protein (IHC) | HPA → |
| Placenta | Trophoblastic cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Cervix | Squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
| Esophagus | Squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
| Oral mucosa | Squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced SFXN3 IHC Tips
Troubleshoot SFXN3 staining in paraffin sections by checking retrieval, compartment, cell identity and controls before comparing signal across samples.
Which retrieval condition should I start with for SFXN3 paraffin-section IHC?
Start with heat-mediated retrieval in EDTA at pH 8.0 for paraffin sections (datasheet A14648-1). The selected tissue image used this condition before 2 μg/ml primary antibody overnight at 4°C (datasheet A14648-1). If staining is weak, check deparaffinisation, heating consistency and section adherence before changing the retrieval condition (standard IHC practice). If the baseline remains weak, test an alternative buffer only as a controlled comparison, with matched sections and the same detection settings (standard IHC practice). Excessive retrieval can damage morphology, making cellular localisation difficult to judge (standard IHC practice).
Could fixation explain weak SFXN3 staining in paraffin sections?
Target-specific fixation sensitivity is unknown: the selected paraffin-section caption does not state a fixative (datasheet A14648-1). Do not infer fixation tolerance from the reported tissue staining pattern or from SFXN3 membrane topology (HPA tissue IHC; UniProt Q9BWM7 topology). Record each specimen’s fixative and processing history, then compare sections processed together using the same EDTA pH 8.0 retrieval (datasheet A14648-1; standard IHC practice). If archived samples differ in handling, include a consistently processed reference section in every run (standard IHC practice). Preserve morphology when judging whether weak staining reflects tissue damage or a detection problem (standard IHC practice).
How should I assess cytoplasmic or nuclear SFXN3 staining?
Assess staining against the expected mitochondrial compartment: SFXN3 is a mitochondrial membrane protein (UniProt Q9BWM7; HPA subcellular). Chromogenic signal may appear cytoplasmic at light-microscope resolution, while HPA tissue IHC also reports cytoplasmic and nuclear expression in most tissues (HPA tissue IHC). Treat predominantly nuclear staining cautiously and compare its distribution with cytoplasmic signal, tissue morphology and an appropriate negative control (HPA tissue IHC; standard IHC practice). High staining is reported in hepatocytes, kidney tubule cells and lung macrophages, which can help orient cell-level review (HPA tissue IHC). A cell-type pattern alone does not establish mitochondrial localisation (standard IHC practice).
How do I troubleshoot SFXN3 staining when the antibody epitope is unclear?
First check whether the antibody’s documented immunogen or epitope is available; the supplied caption does not identify it (datasheet A14648-1). The annotated SFXN3 chain spans residues 1–321, with 4 transmembrane segments and no annotated isoforms (UniProt Q9BWM7). Its transmembrane segments occupy residues 146–164, 174–194, 226–246 and 266–286 (UniProt Q9BWM7 topology). Without an epitope position, do not assume retrieval exposes a particular loop or that absent staining reflects an isoform difference (standard IHC practice). Compare retrieval conditions on adjacent sections while holding antibody concentration and detection constant (standard IHC practice).
What should an IF follow-up test when SFXN3 IHC localisation is uncertain?
Use IF as a separate localisation check: HPA reports enhanced mitochondrial localisation and lists ICC/IF images for SFXN3 (HPA subcellular). Multiplex SFXN3 with a mitochondrial marker and a marker for the cell type being assessed, such as the hepatocytes or macrophages highlighted by tissue IHC (HPA tissue IHC; standard IF practice). Choose spectrally separated fluorophores and evaluate tissue autofluorescence before interpreting apparent overlap, especially where background is bright (standard IF practice). Match permeabilisation to the antibody epitope’s side of the mitochondrial membrane; its position is unspecified here (UniProt Q9BWM7 topology; datasheet A14648-1). Keep the IF workflow and its controls separate from the paraffin-section IHC conditions (standard IF practice).
How can I reduce diffuse or patchy background in SFXN3 chromogenic IHC?
Begin with the documented 10% goat-serum block and 2 μg/ml primary antibody, incubated overnight at 4°C (datasheet A14648-1). The selected image used a peroxidase-conjugated secondary antibody for 30 minutes at 37°C and DAB development (datasheet A14648-1). Include a no-primary control, block endogenous peroxidase and review wash quality when diffuse DAB signal obscures cellular detail (standard IHC practice). Titrate primary antibody and development time on matched sections, changing one condition at a time (standard IHC practice). Judge patchy staining alongside section folds, edges and tissue preservation before attributing it to SFXN3 (standard IHC practice).
How should I score SFXN3 staining across paraffin tissue sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and tissue area before scoring, because HPA reports different staining levels across cell types (HPA tissue IHC). For identifiable cells, record percent positive and intensity, or calculate an H-score from 0–300 using intensity categories 0–3 (standard IHC practice). For dispersed positive cells, report counts per mm² of evaluable tissue and identify the counted cell type (standard IHC practice). Normalise to viable, morphologically comparable tissue or the relevant cell population, rather than total slide area (standard IHC practice). Keep retrieval, detection and scoring criteria consistent, and exclude folds and necrotic regions using predefined rules (standard IHC practice).
What distinguishes credible SFXN3 staining from artefact?
Look for reproducible cellular staining compatible with a mitochondrial membrane protein, while recognising that chromogenic signal may resolve as cytoplasmic (UniProt Q9BWM7; HPA subcellular; standard IHC practice). Check cell identity: HPA reports high staining in hepatocytes, kidney tubule cells and lung macrophages, but no detection in adipocytes (HPA tissue IHC). Predominantly nuclear staining deserves scrutiny because HPA also reports nuclear expression in tissue IHC, whereas its subcellular analysis places SFXN3 in mitochondria (HPA tissue IHC; HPA subcellular). Discount staining confined to section edges, damaged or necrotic areas, or structures positive in a no-primary control (standard IHC practice). Review endogenous peroxidase background before assigning isolated DAB deposits to SFXN3 (standard IHC practice).
Boster reagents
Best SFXN3 / Sideroflexin-3 IHC Antibodies
A14648-1 is listed for human IHC and IF, with images from human paraffin sections for both applications (catalog applications, reactivity, and image captions).
Real IHC data
Anti-SFXN3 Antibody ®
Cat # A14648-1
A14648-1 is listed for human IHC, with images of paraffin sections from human liver cancer, lung adenocarcinoma, spleen, and thyroid cancer tissue (catalog applications, reactivity, and IHC image captions). Its IF image shows a human renal cancer paraffin section (IF image caption).
Which to pick: For tissue IHC, choose A14648-1, a rabbit antibody with human reactivity and documented paraffin-section IHC images (catalog metadata and IHC image captions); the fixative is unreported (IHC image captions). For IF, A14648-1 has a paraffin-section image; ICC validation is unreported (catalog applications and IF image caption). No cross-species option is documented: A14648-1 lists human reactivity only (catalog reactivity).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q9BWM7 (SFXN3_HUMAN, Sideroflexin-3).
- Human Protein Atlas. SFXN3 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. SFXN3 subcellular location (ICC-IF): Localized to the mitochondria..
- Human Protein Atlas. SFXN3 antibody validation summary (2 antibodies).
- Identification of iron metabolism-related genes as prognostic indicators for papillary thyroid carcinoma: a retrospective study. PeerJ 2023 — PMC10290451.
- PubMed PMID:14702039 — UniProt-cited evidence.
- PubMed PMID:15164054 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for SFXN3 (Sideroflexin-3). Expected localisation Tissue IHC shows cytoplasmic and nuclear staining; the protein is annotated at the mitochondrial membrane (HPA tissue IHC; UniProt). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A14648-1). Validated IHC antibody: A14648-1 at 2-5 μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Hippocampus (Neuronal cells); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human liver cancer tissue; fixative not specified (datasheet A14648-1); 2. Fixation: Image fixative and duration unreported (datasheet A14648-1); 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 A14648-1); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A14648-1); 8. Primary antibody: Rabbit anti-SFXN3, 2-5 μg/ml (datasheet A14648-1); 9. Primary incubation: Overnight at 4 °C (datasheet A14648-1). Watch for: Fixation: Keep fixation consistent across paraffin sections. (standard IHC practice; not target-specific); Caveat: Nuclear IHC staining differs from the mitochondrial membrane annotation (HPA tissue IHC; UniProt); Isoform / epitope: No isoforms annotated; epitope orientation is unreported (UniProt); Regulation: Expression regulation is not annotated (UniProt). Evidence: UniProt Q9BWM7, HPA tissue IHC · Approved, HPA ICC-IF · mitochondria., Datasheet A14648-1, PMC10290451. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/SFXN3/immunohistochemistry-ihc-guide.