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RBFOX2 / RNA binding protein fox-1 homolog 2 · IHC design guide
Design Immunohistochemistry for RBFOX2
Plan RBFOX2 IHC in paraffin sections using the catalog antibody’s validated staining conditions. Compare nuclear and cytoplasmic staining with the tissue profile, while accounting for the antibody specificity caution (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
RBFOX2 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 (HPA tissue IHC) | |
| Staining pattern | Nuclear and cytoplasmic signal in diverse cell types (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A05389-2) | |
| Positive control | Caudate+4 more · see all | |
| Negative control | Heart muscle+1 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 | Antibody may detect proteins from more than one gene (HPA tissue IHC) | |
| Regulation | Low tissue specificity reported for RNA expression (HPA tissue RNA) | |
| Isoform / epitope | 10 isoforms; check epitope coverage across variants (UniProt) |
Section 1
Recommended RBFOX2 IHC & IF Protocols
The catalog antibody protocol (datasheet: A05389-2) is followed by published chromogenic IHC protocols for tissue sections (PMC13576891 methods; PMC10730836 methods).
| Sample | Paraffin-embedded human liver cancer tissue; fixative not specified (datasheet A05389-2) |
| Fixation | Image fixative and duration unreported (datasheet A05389-2); 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 A05389-2); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A05389-2) |
| Primary antibody | Rabbit anti-RBFOX2, 2-5 μg/ml (datasheet A05389-2) |
| Primary incubation | Overnight at 4 °C (datasheet A05389-2) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet A05389-2) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | RBFOX2-positive staining in neuronal cells of caudate (HPA tissue IHC: High). HPA tissue profile: General nuclear and cytoplasmic expression. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA pH 8.0 retrieval (datasheet: A05389-2). One published protocol uses CC1 for 32 min without specifying its buffer composition (PMC13576891 methods).
Section 2
What Is the Expected RBFOX2 Staining Pattern?
RBFOX2 should stain nuclei and may stain cytoplasm (UniProt O43251: nucleus and cytoplasm; HPA tissue IHC: general nuclear and cytoplasmic expression). Expect stronger staining in HPA High cell populations, including neuronal cells in caudate and glomerular cells in kidney (HPA tissue IHC). HPA rates the tissue IHC profile Approved, with medium consistency against RNA data and a warning that the antibodies may recognize proteins from more than one gene (HPA tissue IHC). RBFOX2 has no transmembrane segment (UniProt O43251 topology).
| Nuclear staining, with or without cytoplasmic staining, in the expected cell population. | This fits the reported compartments (UniProt O43251: nucleus and cytoplasm; HPA tissue IHC: general nuclear and cytoplasmic expression). For a tissue check, compare the named cell population rather than judging every cell in the section as equally positive (HPA tissue IHC: cell-specific levels). |
| Staining appears chiefly at the cell membrane or extracellularly. | That is outside the reported nuclear and cytoplasmic pattern (UniProt O43251; HPA tissue IHC). RBFOX2 has no transmembrane segment (UniProt O43251 topology). Review morphology and detection background before treating this distribution as RBFOX2. |
| A cell population reported as Not detected stains strongly. | Cardiomyocytes in heart muscle and glandular cells in parathyroid are reported as Not detected (HPA tissue IHC). Strong staining there warrants checking cell identity, antibody specificity and endogenous detection activity; the HPA profile cautions that antibodies may recognize proteins from more than one gene (HPA tissue IHC). |
| Chromogen covers cells broadly without a clear nuclear or cytoplasmic pattern. | Diffuse staining cannot establish the reported RBFOX2 distribution (UniProt O43251; HPA tissue IHC). Compare a no-primary control and inspect tissue edges and counterstain; nonspecific binding or detection background can obscure compartments (standard IHC practice). |
| No staining appears in an expected positive cell population. | Check that the scored cells match the reference: HPA reports High staining in caudate neuronal cells, kidney glomerular cells and lung alveolar cells (HPA tissue IHC). A blank result in such cells prompts assay troubleshooting; it alone does not establish absent RBFOX2. |
💡Expected RBFOX2 appearanceCall a result consistent with RBFOX2 when the expected cells show discernible nuclear staining, possibly with cytoplasmic staining, including High populations such as caudate neurons (UniProt O43251; HPA tissue IHC); strong membrane, extracellular or indiscriminate staining is outside that reported pattern (UniProt O43251 topology; HPA tissue IHC).
| Cell population and tissue | HPA reports High staining in caudate neurons, endometrial stromal cells, kidney glomerular cells and ovarian stromal cells, but Not detected in cardiomyocytes and parathyroid glandular cells (HPA tissue IHC). Score the named cells, since whole-section intensity can conceal that distinction. |
| Antibody validation | The tissue IHC profile is Approved, with medium staining–RNA consistency and a warning that antibodies may recognize proteins from more than one gene (HPA tissue IHC). HPA006240 and HPA075862 have IHC Approved status; this does not establish specificity for the catalog antibody (HPA antibodies). |
| Compartment | UniProt reports nuclear and cytoplasmic localization, while HPA tissue IHC describes general nuclear and cytoplasmic expression (UniProt O43251; HPA tissue IHC). HPA ICC-IF supports nucleoplasm as the main location (HPA subcellular); use that observation as an IF comparison, not an IHC intensity rule. |
| Isoforms and antibody coverage | UniProt lists 10 RBFOX2 isoforms (UniProt O43251). The payload gives no epitope map or isoform coverage for the catalog antibody, so a staining difference cannot be assigned to a particular isoform from these records. |
| Target-specific fixation sensitivity | Neither supplied source reports how fixation changes RBFOX2 staining (UniProt O43251; HPA tissue IHC). Record fixation and retrieval conditions during optimization, but do not infer an RBFOX2-specific fixation effect from tissue intensity or topology. |
| Situation | Likely cause | Next action |
|---|---|---|
| Expected positive cells are blank. | Cell identity, antibody performance or detection may be at issue; HPA reports High staining in caudate neuronal cells and kidney glomerular cells (HPA tissue IHC). | Verify the scored cell population and run an appropriate positive section with the same IHC conditions. Check primary incubation, retrieval and detection steps systematically (standard IHC practice); the payload supplies no RBFOX2-specific retrieval condition. |
| Signal is diffuse across the section. | Nonspecific binding or chromogenic detection background can obscure cell boundaries (standard IHC practice). The expected distribution is nuclear and cytoplasmic (UniProt O43251; HPA tissue IHC). | Compare a no-primary control, inspect morphology, and optimize blocking, washing and primary-antibody concentration one variable at a time (standard IHC practice). |
| Membrane or extracellular staining dominates. | This conflicts with the reported nuclear and cytoplasmic pattern and lack of a transmembrane segment (UniProt O43251; HPA tissue IHC). The image alone does not identify the source of staining. | Check a no-primary control and review whether staining follows cells or section artefacts. Reassess antibody specificity before scoring that signal as RBFOX2 (standard IHC practice; HPA tissue IHC: antibody caveat). |
| Cardiomyocytes stain strongly. | HPA reports RBFOX2 as Not detected in cardiomyocytes and warns that tissue antibodies may recognize proteins from more than one gene (HPA tissue IHC). Endogenous detection activity is another assay possibility (standard IHC practice). | Confirm the cells are cardiomyocytes; compare no-primary and positive-section controls. Investigate background and specificity before reporting a new tissue pattern (standard IHC practice). |
| High and low regions look similar. | The named populations differ: lung alveolar cells are High, while bronchus respiratory epithelial cells are Low (HPA tissue IHC). Broad chromogen deposits can make intensity comparisons unreliable (standard IHC practice). | Score the specified cells separately using consistent exposure to chromogen and counterstain. Review controls and reduce excessive background before interpreting intensity (standard IHC practice). |
| Does IF/ICC need the same protocol or readout? | HPA supports nucleoplasmic localization in ICC-IF, whereas its tissue IHC profile reports general nuclear and cytoplasmic expression (HPA subcellular; HPA tissue IHC). | Use nucleoplasmic signal as the IF/ICC localization comparison (HPA subcellular). Consult the separate IF/ICC guide for its workflow; this IHC section supplies no IF/ICC protocol. |
Experiment evidence
IHC & IF images showing RBFOX2 staining
A05389-2 · IHCIHC analysis of FOX2/RBM9/RBFOX2 using anti-FOX2/RBM9/RBFOX2 antibody (A05389-2). FOX2/RBM9/RBFOX2 was detected in a paraffin-embedded secti
A05389-2 · IHCIHC analysis of FOX2/RBM9/RBFOX2 using anti-FOX2/RBM9/RBFOX2 antibody (A05389-2). FOX2/RBM9/RBFOX2 was detected in a paraffin-embedded secti
A05389-2 · IFIF analysis of FOX2/RBM9/RBFOX2 using anti-FOX2/RBM9/RBFOX2 antibody (A05389-2). FOX2/RBM9/RBFOX2 was detected in a paraffin-embedded sectio
A05389-1 · IHCIHC analysis of RBFOX2 using anti-RBFOX2 antibody (A05389-1). RBFOX2 was detected in a paraffin-embedded section of human endometrial cancer
A05389-1 · IHCIHC analysis of RBFOX2 using anti-RBFOX2 antibody (A05389-1). RBFOX2 was detected in a paraffin-embedded section of mouse brain tissue. Heat
A05389-1 · IFIF analysis of RBFOX2 using anti-RBFOX2 antibody (A05389-1) and anti-Tubulin Alpha antibody (M03989-3). RBFOX2 was detected in immunocytoche
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000100320-RBFOX2/tissue/cerebral+cortex#imid_1869096
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000100320-RBFOX2/tissue/colon#imid_1869030
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000100320-RBFOX2/tissue/liver#imid_1869031
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000100320-RBFOX2/tissue/kidney#imid_1869032
HPA · ICC-IF SH-SY5YHuman Protein Atlas immunofluorescence, SH-SY5Y, antibody HPA006455; green = RBFOX2, blue = nucleus, red = microtubules.
HPA · ICC-IF SH-SY5YHuman Protein Atlas immunofluorescence, SH-SY5Y, antibody HPA006455; green = RBFOX2, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for RBFOX2 IHC & IF
🧪Run caudate first: neuronal cells should stain (HPA: High in caudate neuronal cells). Run heart muscle as the negative tissue because cardiomyocytes are not detected (HPA: Not detected in cardiomyocytes); on the caudate slide, use cells without nuclear signal as an internal background reference, without assuming that every non-neuronal cell is negative (HPA: High in neuronal cells; standard IHC practice).
Positive control tissue: Caudate (Neuronal cells, HPA High)
Negative control tissue: Heart muscle (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show RBFOX2 in SH-SY5Y, SiHa, U2OS, with annotated localisation: Nucleoplasm (supported) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control, a host- and clonality-matched rabbit IgG control, and a biological negative such as RBFOX2 knockout material or cognate-peptide blocking if the immunogen is available (selected-SKU caption: rabbit primary; standard IHC control practice). Block endogenous peroxidase for DAB detection, and assess brain pigment as a possible source of misleading signal in caudate (standard IHC practice).
⚠️Feasibility: No target-specific fixation window or fixation effect is reported in the supplied evidence; the fixative is unreported in the A05389-2 paraffin-section caption (selected-SKU caption). That caption uses heat-mediated EDTA retrieval at pH 8.0 in liver cancer tissue, so retrieval conditions need checking in caudate (selected-SKU caption; standard IHC practice). The supplied evidence does not establish that frozen sections or IF are easier; brain autofluorescence may complicate IF interpretation (HPA: ICC-IF nucleoplasmic localization; standard IF practice).
HPA tissue IHC evidence for RBFOX2
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Medium consistency between antibody staining and RNA expression data. Caution, targets protein from more than one gene.). 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 |
|---|---|---|---|---|
| Caudate | Neuronal cells | High | Protein (IHC) | HPA → |
| Cerebellum | Cells in molecular layer | High | Protein (IHC) | HPA → |
| Cerebral cortex | Neuronal cells | High | Protein (IHC) | HPA → |
| Endometrium | Cells in endometrial stroma | High | Protein (IHC) | HPA → |
| Hippocampus | Neuronal cells | High | Protein (IHC) | HPA → |
Section 3
Advanced RBFOX2 IHC Tips
Use the IHC-validated antibody's paraffin-section result as a starting point, then check compartment, cell type and controls when interpreting RBFOX2 staining.
Which retrieval conditions should I try first for RBFOX2 in paraffin sections?
Use heat-mediated antigen retrieval in EDTA at pH 8.0 before chromogenic staining of paraffin sections (datasheet A05389-2). This is the retrieval used for the selected antibody's human liver cancer section, followed by 2 μg/ml primary antibody overnight at 4°C (caption A05389-2). If nuclear signal is weak, compare retrieval duration across adjacent sections while holding antibody concentration, detection and imaging conditions constant; record tissue damage as well as staining. Judge improvement in identifiable nuclei and expected cell populations, since RBFOX2 has a supported nucleoplasmic location and HPA reports general nuclear and cytoplasmic tissue staining (HPA subcellular; HPA tissue IHC).
How should I troubleshoot inconsistent RBFOX2 staining after fixation?
The selected paraffin-section caption does not state a fixative, so RBFOX2-specific fixation sensitivity remains unknown (caption A05389-2). Record each specimen's fixative, fixation duration, processing schedule and section age, then compare sections processed alike before attributing differences to RBFOX2 biology. Start with the documented EDTA retrieval at pH 8.0 and 2 μg/ml primary incubation overnight at 4°C, changing one variable per comparison (caption A05389-2). Include a reference section with identifiable nuclear staining, because RBFOX2 is supported in the nucleoplasm and variable processing can make a weak result difficult to interpret (HPA subcellular).
Should RBFOX2 staining be nuclear, cytoplasmic, or both?
Prioritise nucleoplasmic staining when identifying RBFOX2-positive cells, because its supported subcellular location is the nucleoplasm (HPA subcellular). Cytoplasmic signal can occur: UniProt lists nucleus and cytoplasm, and HPA describes general nuclear and cytoplasmic expression in tissue IHC (UniProt O43251; HPA tissue IHC). Compare nuclear and cytoplasmic staining separately on the same section, using a nuclear counterstain and consistent exposure or microscope settings across samples. Treat staining confined to extracellular material or a crisp membrane rim cautiously, since RBFOX2 has no transmembrane segment and is an RNA-binding splicing regulator (UniProt O43251 topology; UniProt O43251 function).
Could isoforms or epitope changes explain different RBFOX2 IHC patterns?
RBFOX2 has 10 listed isoforms and an RNA recognition motif spanning residues 121–197, so epitope position matters when comparing antibodies (UniProt O43251). The supplied caption does not identify the catalog antibody's epitope; do not assign an isoform-specific interpretation to its staining (caption A05389-2). UniProt also lists modified arginine residues, including positions 281, 297 and 329, without establishing an effect on this antibody (UniProt O43251). If antibodies disagree, compare their documented immunogens, stain matched serial sections under each validated condition, and require an independent specificity control before attributing discordance to an isoform or modification.
How can IF help verify RBFOX2 localisation and cell identity?
For secondary IF or ICC work, pair RBFOX2 with a marker for the cell population under study; HPA reports high staining in neuronal cells in caudate and cerebral cortex (HPA tissue IHC). Choose well-separated fluorophores and consider a far-red RBFOX2 channel when tissue autofluorescence obscures shorter-wavelength signal; check single-label and unstained controls before judging colocalisation. Use permeabilisation sufficient for antibody access to the nucleus, because RBFOX2 has no transmembrane segment and its supported location is nucleoplasmic (UniProt O43251 topology; HPA subcellular). HPA lists ICC/IF images in 3 cell lines, but the supplied A05389-2 caption documents chromogenic tissue IHC, not IF validation of that antibody (HPA subcellular; caption A05389-2).
What should I change if RBFOX2 DAB staining is diffuse?
Separate diffuse cellular staining from background by comparing nuclei, cytoplasm and blank spaces within the same section; HPA reports general nuclear and cytoplasmic staining (HPA tissue IHC). The documented workflow used 10% goat serum, 2 μg/ml primary antibody overnight at 4°C, and DAB development (caption A05389-2). Include a no-primary control, check the routine endogenous peroxidase block, and shorten DAB development if pigment accumulates outside cells. If background persists, compare lower primary concentrations on adjacent sections while retaining the EDTA pH 8.0 retrieval, and judge signal against expected nucleoplasmic staining (datasheet A05389-2; HPA subcellular).
How should I score RBFOX2 across stained tissue sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring: RBFOX2 has supported nucleoplasmic localisation, while tissue IHC also shows cytoplasmic expression (HPA subcellular; HPA tissue IHC). Report the percentage of positive nuclei and an H-score using intensity categories 0–3, or count positive cells per mm² when cell density is the question. Normalise positive counts to all evaluable cells of the specified population, or to measured viable tissue area for density; keep retrieval, antibody concentration and DAB development matched. Record nuclear and cytoplasmic results separately, and exclude folds, necrosis and edges from the measured area so technical staining does not inflate the score.
How do I distinguish genuine RBFOX2 signal from IHC artefact?
A credible result places signal in identifiable cells, especially their nuclei, consistent with supported nucleoplasmic RBFOX2 localisation (HPA subcellular). Compare the questioned area with reference populations: HPA reports high staining in caudate neuronal cells and kidney glomerular cells, but no detected staining in heart muscle cardiomyocytes (HPA tissue IHC). Be cautious with staining restricted to tissue edges, necrosis, extracellular deposits or a membrane outline; RBFOX2 has no transmembrane segment (UniProt O43251 topology). Check a no-primary control for endogenous enzyme or detection background, and avoid treating one positive section as definitive specificity evidence because HPA warns that its tissue antibody targets protein from more than one gene (HPA tissue IHC).
Boster reagents
Best RBFOX2 / RNA binding protein fox-1 homolog 2 IHC Antibodies
Rabbit anti-RBFOX2 antibodies have IHC images from human paraffin sections and mouse and rat brain, plus IF images from human tissue and cells and mouse and rat brain (catalog image captions).
Real IHC data
Anti-FOX2/RBM9/RBFOX2 Antibody ®
Cat # A05389-2
Real IHC data
Anti-Fox2/RBM9/RBFOX2 Antibody ®
Cat # A05389-1
A05389-1 has IHC images from human endometrial cancer and mouse and rat brain, plus IF images from U2OS cells and mouse and rat brain (A05389-1 image captions). A05389-2 has IHC images from human liver cancer, esophageal squamous carcinoma, lung cancer and cortical adenoma, plus an IF image from human lung cancer (A05389-2 image captions).
Which to pick: For human tissue IHC, choose A05389-2 when its paraffin-section examples fit your study; its IHC caption reports EDTA retrieval at pH 8.0 and 2 μg/ml primary antibody overnight at 4°C, while the fixative is unreported (A05389-2 IHC captions). Choose A05389-1 for IF/ICC or cross-species tissue IHC: ICC is listed, and its images show human cell IF and mouse and rat brain IHC; its own IHC captions report EDTA retrieval at pH 8.0 and 2 μg/ml primary antibody overnight at 4°C, with fixative unreported (A05389-1 applications; A05389-1 IF and IHC captions). Both have rabbit hosts, and clonality is unreported (catalog host and clone fields).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry O43251 (RFOX2_HUMAN, RNA binding protein fox-1 homolog 2).
- Human Protein Atlas. RBFOX2 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. RBFOX2 subcellular location (ICC-IF): Localized to the nucleoplasm..
- Human Protein Atlas. RBFOX2 antibody validation summary (3 antibodies).
- Functional validation of driver mutation-specific uveal melanoma biomarkers: role of COL9A3 in cancer cell plasticity. The Journal of pathology 2026 — PMC13576891.
- Novel Rbfox2 isoforms associated with alternative exon usage in rat cortex and suprachiasmatic nucleus. Scientific reports 2017 — PMC5577181.
- RBFOX2 deregulation promotes pancreatic cancer progression and metastasis through alternative splicing. Nature communications 2023 — PMC10730836.
- The effect of Rbfox2 modulation on retinal transcriptome and visual function. Scientific reports 2020 — PMC7665016.
- PubMed PMID:11875103 — UniProt-cited evidence.
- PubMed PMID:12529303 — UniProt-cited evidence.
- PubMed PMID:15461802 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for RBFOX2 (RNA binding protein fox-1 homolog 2). Expected localisation Nuclear and cytoplasmic staining (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A05389-2). Validated IHC antibody: A05389-2 at 2-5 μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Caudate (Neuronal cells); negative Heart muscle. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human liver cancer tissue; fixative not specified (datasheet A05389-2); 2. Fixation: Image fixative and duration unreported (datasheet A05389-2); 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 A05389-2); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A05389-2); 8. Primary antibody: Rabbit anti-RBFOX2, 2-5 μg/ml (datasheet A05389-2); 9. Primary incubation: Overnight at 4 °C (datasheet A05389-2). Watch for: Fixation: Keep fixation consistent across paraffin sections. (standard IHC practice; not target-specific); Caveat: Antibody may detect proteins from more than one gene (HPA tissue IHC); Isoform / epitope: 10 isoforms; check epitope coverage across variants (UniProt); Regulation: Low tissue specificity reported for RNA expression (HPA tissue RNA). Evidence: UniProt O43251, HPA tissue IHC · Approved, HPA ICC-IF · nucleoplasm., Datasheet A05389-2, PMC13576891, PMC10730836. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/RBFOX2/immunohistochemistry-ihc-guide.