RBFOX3 / RNA binding protein fox-1 homolog 3 · IHC design guide

Design Immunohistochemistry for RBFOX3

Plan chromogenic RBFOX3 IHC in paraffin sections using nuclear staining in cerebellar granular cells as a positive reference (HPA tissue IHC). This guide covers the catalog antibody at 1:50 (datasheet M11954) and the risk of off-target staining (HPA tissue IHC).

Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use

Immunohistochemistry protocol sheet for RBFOX3 (IHC for RBFOX3): expected localisation Predominantly neuronal nuclei; cytoplasmic signal is possible (UniProt), antibody M11954, validated IHC image, and IHC protocol steps — Printable **RBFOX3** IHC protocol sheet — expected localisation Predominantly neuronal nuclei; cytoplasmic signal is possible (UniProt), antibody M11954, controls and protocol steps. Open the full RBFOX3 IHC guide →

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RBFOX3 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	Predominantly neuronal nuclei; cytoplasmic signal is possible (UniProt)
	Staining pattern	Strong nuclear staining in cerebellar granular cells (HPA tissue IHC)
	Antigen retrieval	EDTA pH 8.0 HIER, heat-mediated (datasheet M11954)
	Positive control ⓘ	Cerebellum+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 paraffin-section fixation consistent (standard IHC practice; not target-specific)
	Caveat	Presumed off-target binding may confound staining (HPA tissue IHC)
	Regulation	Expression regulation unreported (UniProt)
	Isoform / epitope	2 isoforms; epitope coverage is unknown (UniProt)

Section 1

Recommended RBFOX3 IHC & IF Protocols

The catalog antibody has a datasheet IHC-P protocol (datasheet: M11954). The published IHC protocols below cover human brain, piglet neocortex, and caudate putamen and substantia nigra (PMC4665621; PMC10605428; PMC9826750).

Recommended immunohistochemistry (IHC-P) protocol parameters
Sample	Paraffin-embedded mouse brain tissue; fixative not specified (datasheet M11954)
Fixation	Image fixative and duration unreported (datasheet M11954); 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 M11954); 20 min, 95–100 °C (standard)
Peroxidase block	3% H2O2, 10 min, room temperature (standard)
Blocking	10% goat serum (datasheet M11954)
Primary antibody	Rabbit monoclonal (clone AO-18) anti-RBFOX3, 1:50 (datasheet M11954)
Primary incubation	Overnight at 4 °C (datasheet M11954)
Detection	HRP-conjugated secondary, DAB chromogen (datasheet M11954)
Counterstain	Hematoxylin, blue, dehydrate and mount (standard)
Expected result	RBFOX3-positive staining in granular cells - nucleus of cerebellum (HPA tissue IHC: High). HPA tissue profile: Distinct expression in CNS. No signal in the no-primary control.

💡Decision noteStart with heat-mediated EDTA pH 8.0 retrieval for the catalog antibody (datasheet: M11954); the human brain protocol used Tris-EDTA pH 9.0 (PMC4665621).

Section 2

What Is the Expected RBFOX3 Staining Pattern?

RBFOX3 staining should concentrate in neuronal nuclei: UniProt describes it as largely restricted to neuronal nuclei, and HPA reports high staining in cerebellar granular cell nuclei (UniProt A6NFN3; HPA tissue IHC). HPA rates the tissue pattern “Enhanced” but notes medium agreement with RNA and presumed off-target binding; interpret unexpected staining cautiously (HPA tissue IHC). RBFOX3 has no transmembrane segment (UniProt A6NFN3 topology).

What am I looking at on my slide?
Distinct nuclear staining in cerebellar granular cells; neuronal staining in cerebral cortex.	This matches HPA's high cerebellar granular cell nuclear and cerebral cortical neuronal staining. Score the nuclear pattern in its cell context; the cortex entry does not independently specify a compartment (HPA tissue IHC; UniProt A6NFN3 localization).
Predominantly cytoplasmic staining in neurons, without a clear nuclear pattern.	Review compartment assignment and controls: RBFOX3 is largely nuclear, although UniProt reports significant neuronal cytoplasmic localization in brains from HIV-infected individuals with cognitive impairment. Cytoplasmic staining alone is therefore context dependent, not automatic proof of an artefact (UniProt A6NFN3 localization).
Strong staining in cells outside the expected neuronal population.	Check cell identity and detection controls before calling it RBFOX3. HPA notes presumed off-target binding and cautions that staining can reflect protein from more than one gene. A positive signal alone cannot establish specificity in an unexpected cell type (HPA tissue IHC).
Diffuse color across cells, stroma, or a broad tissue area.	Assess the section alongside a no-primary control; widespread color without cell-specific nuclear enrichment can arise from nonspecific binding or endogenous detection activity (general IHC practice). HPA's tissue profile alone cannot assign the cause of background on this slide (HPA tissue IHC).
No signal in cerebellar granular cells or cerebral cortical neurons.	A blank result conflicts with HPA's high staining in these populations. First confirm the relevant cells are present, then review the IHC-validated antibody, detection run, and controls; absence on one section does not establish absent RBFOX3 expression (HPA tissue IHC; general IHC practice).

💡Expected RBFOX3 appearanceA convincing positive is distinct neuronal nuclear staining, especially high cerebellar granular cell nuclear staining; diffuse or unexpected cell staining needs control-based review (HPA tissue IHC; UniProt A6NFN3 localization).

How each factor affects the staining
Anatomic and cellular context	HPA reports high cerebellar granular cell nuclear and cerebral cortical neuronal staining, with medium staining in caudate and hippocampal neurons. Compare like cell populations when judging signal strength (HPA tissue IHC).
Specificity of the tissue pattern	HPA calls the profile “Enhanced” while reporting medium staining–RNA consistency and presumed off-target binding. Its colon enterocyte result is medium; do not treat every non-neuronal positive as independently confirmed RBFOX3 (HPA tissue IHC).
Antibody-specific validation	The HPA030790 IHC result is “Enhanced”; HPA075862 and CAB078782 are “Supported.” These labels describe HPA validation for those antibodies and cannot be transferred to the catalog antibody without its own evidence (HPA antibody validation).
Compartment and protein forms	UniProt lists nuclear and cytoplasmic localization, two isoforms, and a full-length 1–312 chain with no signal peptide, propeptide, or transmembrane segment. No epitope position is supplied, so isoform-dependent IHC detection cannot be predicted here (UniProt A6NFN3).
IF/ICC Q: What localization is reported?	A: HPA reports supported nucleoplasmic localization in ICC-IF, with images from A-549, U-251MG, and U2OS. That observation is a compartment reference; it does not replace tissue IHC controls or establish an IF protocol (HPA subcellular ICC-IF).

Why is my staining missing, weak or wrong?
Situation	Likely cause	Next action
Known-positive neuronal nuclei are blank.	The expected population may be absent from the section, or the staining run may have failed; HPA reports high signal in cerebellar granular cells and cortical neurons (HPA tissue IHC; general IHC practice).	Locate the relevant cells on the counterstain, inspect the positive control, then review the IHC-validated antibody and detection steps (general IHC practice).
Neurons appear cytoplasmic with little nuclear signal.	The pattern differs from the usual neuronal nuclear localization; UniProt also records a specific HIV-associated brain context with significant cytoplasmic localization (UniProt A6NFN3 localization).	Verify nuclear boundaries and compare a neuronal positive control. Document the specimen context before treating cytoplasmic staining as either genuine relocalization or artefact (general IHC practice; UniProt A6NFN3).
Unexpected non-neuronal cells stain strongly.	The signal may be off-target or detection-related; HPA explicitly notes presumed off-target binding. HPA also records medium colon enterocyte staining, so location alone cannot settle specificity (HPA tissue IHC).	Compare tissue morphology and no-primary controls; seek antibody-specific corroboration before assigning an unexpected positive cell population to RBFOX3 (HPA tissue IHC; general IHC practice).
Diffuse brown staining obscures nuclei.	Nonspecific binding or endogenous enzyme activity can contribute to chromogenic background (general IHC practice). HPA tissue levels do not identify the mechanism in an individual section (HPA tissue IHC).	Inspect a no-primary control and the detection reagents; adjust blocking, washes, or endogenous enzyme quenching according to the control result (general IHC practice).
Weak signal in caudate or hippocampus seems inconsistent with stronger cerebellum.	HPA reports medium neuronal staining in caudate and hippocampus versus high cerebellar granular cell nuclear staining; different recorded levels need not indicate a failed run (HPA tissue IHC).	Score each region against its own expected cell population and include a strong neuronal reference section in the run (HPA tissue IHC; general IHC practice).
Low non-neuronal staining is mistaken for a definitive positive.	HPA lists low staining in several non-neuronal populations and warns of presumed off-target binding; low signal is especially hard to interpret without controls (HPA tissue IHC).	Record cell type, compartment, and control behavior; reserve a positive RBFOX3 interpretation for a supported pattern or further antibody-specific corroboration (HPA tissue IHC; general IHC practice).

Source: UniProt A6NFN3 Human Protein Atlas tissue Human Protein Atlas subcellular

Experiment evidence

IHC & IF images showing RBFOX3 staining

IHC analysis of NeuN using anti-NeuN antibody (M11954). NeuN was detected in a paraffin-embedded section of mouse brain tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1:50 rabbit anti-NeuN Antibody (M11954) overnight at 4°C. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using HRP Conjugated Rabbit IgG Super Vision Assay Kit (Catalog # SV0002) with DAB as the chromogen.

M11954 · IHCIHC analysis of NeuN using anti-NeuN antibody (M11954). NeuN was detected in a paraffin-embedded section of mouse brain tissue. Heat mediate

IHC analysis of NeuN using anti-NeuN antibody (M11954). NeuN was detected in a paraffin-embedded section of mouse cerebellum tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1:50 rabbit anti-NeuN Antibody (M11954) overnight at 4°C. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using HRP Conjugated Rabbit IgG Super Vision Assay Kit (Catalog # SV0002) with DAB as the chromogen.

M11954 · IHCIHC analysis of NeuN using anti-NeuN antibody (M11954). NeuN was detected in a paraffin-embedded section of mouse cerebellum tissue. Heat me

HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000167281-RBFOX3/tissue/cerebral+cortex#imid_8139345

HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000167281-RBFOX3/tissue/colon#imid_8138918

HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000167281-RBFOX3/tissue/liver#imid_8138919

HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000167281-RBFOX3/tissue/kidney#imid_8138912

HPA · ICC-IF A-549Human Protein Atlas immunofluorescence, A-549, antibody HPA030790; green = RBFOX3, blue = nucleus, red = microtubules.

← swipe / scroll for more images →

Real IHC / IF data behind the staining-pattern expectations in this section.

Sample controls for RBFOX3 IHC & IF

🧪Run cerebellum first: granular cell nuclei should stain strongly (HPA: High in cerebellar granular cell nuclei). Use adipose tissue as the negative tissue (HPA: Not detected in adipocytes); neighboring non-neuronal nuclei on the cerebellar slide should lack specific nuclear staining (UniProt A6NFN3: largely restricted to neuronal nuclei).

Positive control tissue: Cerebellum (Granular cells - nucleus, HPA High)

Negative control tissue: Adipose tissue (HPA Not detected)

ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show RBFOX3 in A-549, U-251MG, U2OS, with annotated localisation: Nucleoplasm (supported) (HPA subcellular).

Technical controls: Include a no-primary, secondary-only control, an isotype-matched nonimmune rabbit IgG control, and RBFOX3 knockout tissue as a biological negative (selected M11954 caption: rabbit primary; standard IHC controls). Quench endogenous peroxidase in brain sections before chromogenic detection (standard IHC practice).

⚠️Feasibility: The selected M11954 paraffin-section caption reports heat retrieval in EDTA at pH 8.0, but does not establish that retrieval is required or report the fixative (selected M11954 caption). A target-specific fixation window or fixation effect is unreported, and the supplied evidence does not establish that frozen sections or IF/ICC are easier (supplied target/application evidence). Brain endogenous peroxidase can cause chromogenic background, so assess staining against the controls (standard IHC practice).

HPA tissue IHC evidence for RBFOX3

Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — Medium consistency between antibody staining and RNA expression data. Caution, targets protein from more than one gene. Presumed off target binding observed and disregarded.). 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
Cerebellum	Granular cells - nucleus	High	Protein (IHC)	HPA →
Cerebral cortex	Neuronal cells	High	Protein (IHC)	HPA →
Caudate	Neuronal cells	Medium	Protein (IHC)	HPA →
Colon	Enterocytes	Medium	Protein (IHC)	HPA →
Hippocampus	Neuronal cells	Medium	Protein (IHC)	HPA →

Undetected expression · recommended negative controls

Tissue	Cell type	Level	Evidence	Source
Adipose tissue	Adipocytes	Not detected	Protein (IHC)	HPA →
Adrenal gland	Glandular cells	Not detected	Protein (IHC)	HPA →
Appendix	Endocrine cells	Not detected	Protein (IHC)	HPA →
Bone marrow	Hematopoietic cells	Not detected	Protein (IHC)	HPA →
Breast	Adipocytes	Not detected	Protein (IHC)	HPA →

Section 3

Advanced RBFOX3 IHC Tips

Troubleshoot RBFOX3 staining in paraffin sections by checking retrieval, nuclear localisation and neuronal cell identity alongside appropriate controls.

How should I retrieve RBFOX3 when nuclear staining is weak?

Start with heat-mediated retrieval in EDTA at pH 8.0 for paraffin sections (datasheet M11954). The demonstrated workflow then uses the catalog antibody at 1:50 overnight at 4°C, so keep those conditions consistent while assessing retrieval (datasheet M11954). Compare a well-stained neuronal region and a no-primary control on the same run; uneven heating or tissue loss can resemble weak antigen detection (standard IHC practice). If staining remains weak, adjust heating duration on matched sections before testing another buffer, and judge improvement by nuclear signal in granular cells rather than stronger diffuse background (HPA: high nuclear staining in cerebellar granular cells; standard IHC practice).

Could fixation explain inconsistent RBFOX3 staining between paraffin blocks?

Target-specific RBFOX3 sensitivity to fixation is unknown here: the selected paraffin-section caption does not report a fixative or fixation duration (datasheet M11954). Record the fixative, time to fixation, duration and processing history for each block, because these variables can change antigen accessibility and morphology in routine IHC (standard IHC practice). Compare blocks using the same EDTA pH 8.0 retrieval and 1:50 primary incubation before attributing differences to fixation (datasheet M11954; standard IHC practice). Include a neuronal internal control in each section, and interpret lost nuclear staining only alongside preserved tissue architecture and control performance (HPA: nuclear staining in cerebellar granular cells; standard IHC practice).

Should cytoplasmic RBFOX3 staining count as positive in brain sections?

Prioritise well-defined neuronal nuclear staining when judging routine chromogenic sections, consistent with the supported nucleoplasmic location and strong nuclear signal in cerebellar granular cells (HPA subcellular: nucleoplasm supported; HPA tissue: cerebellum). RBFOX3 can also occur in cytoplasm, and substantial neuronal cytoplasmic localisation has been reported in brains from HIV-infected individuals with cognitive impairment (UniProt A6NFN3: subcellular location). Score nuclear and cytoplasmic staining separately rather than combining them into one positive-cell count (standard IHC practice). Compare the pattern with adjacent neuronal morphology and a no-primary control; diffuse colour across cell types or section edges warrants investigation before a biological interpretation (standard IHC practice).

Could isoforms or epitope masking change the RBFOX3 IHC pattern?

RBFOX3 has 2 annotated isoforms and an RNA recognition motif at residues 100–175, but the supplied product evidence does not map this antibody’s epitope (UniProt A6NFN3: isoforms and domains; datasheet M11954). Arginine methylation is annotated at residues 223 and 272; whether either modification affects this antibody’s staining is unknown (UniProt A6NFN3: modified residues; datasheet M11954). Keep retrieval at EDTA pH 8.0 while comparing matched sections, so differences in signal are easier to assess (datasheet M11954; standard IHC practice). Do not assign an isoform or modification from chromogenic staining alone; an epitope-mapped reagent and an independent method would be needed for that conclusion (standard IHC practice).

How can I check RBFOX3 localisation by multiplex IF?

Pair RBFOX3 with a marker for the expected neuronal cell type and a nuclear counterstain, then inspect overlap cell by cell rather than relying on tissue-level fluorescence (HPA tissue: neuronal staining in cerebral cortex; standard IF practice). Choose a well-separated, preferably far-red fluorophore for the weaker signal and image an unstained section to assess tissue autofluorescence (standard IF practice). RBFOX3 has no transmembrane segment and is chiefly nuclear, so permeabilise cells sufficiently to reach intracellular and nuclear epitopes while checking that morphology remains intact (UniProt A6NFN3: topology and subcellular location; standard IF practice). Optimise IF fixation and permeabilisation independently; the 1:50 overnight condition documents paraffin-section IHC, not an IF validation (datasheet M11954).

What should I change when DAB obscures neuronal nuclei?

First compare the stained section with a no-primary control and review the duration of DAB development; colour appearing in both sections points to a detection or tissue-background problem (standard IHC practice). The documented workflow uses 10% goat-serum blocking, 1:50 primary antibody overnight at 4°C, and a peroxidase-linked secondary before DAB (datasheet M11954). Include an endogenous peroxidase block and wash thoroughly between steps, then shorten DAB development if nuclear detail is lost (standard IHC practice). Recheck background against expected neuronal nuclear staining; broad staining of unrelated compartments should not be rescued by simply increasing primary concentration (HPA subcellular: nucleoplasm supported; standard IHC practice).

How should I quantify RBFOX3-positive cells across sections? ⚠ ANSWER MARKED FOR VERIFICATION

Define the anatomical region and count cells with clearly resolved nuclear DAB signal, using a consistent threshold and blinded review where possible (HPA subcellular: nucleoplasm supported; standard IHC practice). Report the percentage of positive neuronal cells relative to all evaluable neuronal cells, or positive-cell density per mm² of analysed tissue; state which denominator was used (standard IHC practice). If staining intensity is relevant, an H-score from 0–300 can combine the percentages at 0–3 intensity levels, but score nuclear and cytoplasmic compartments separately (standard IHC practice; UniProt A6NFN3: subcellular location). Exclude folds, damaged edges and poorly preserved regions, and keep section thickness, retrieval and imaging settings consistent across groups (standard IHC practice).

How can I distinguish true RBFOX3 staining from artefact?

A convincing result shows discrete signal in cells with neuronal morphology, usually centred in nuclei, as supported by the nucleoplasmic annotation and cerebral and cerebellar tissue patterns (HPA subcellular: nucleoplasm supported; HPA tissue: cerebral cortex and cerebellum). Check any unusual cytoplasmic signal separately because neuronal cytoplasmic localisation is documented in a specific disease context (UniProt A6NFN3: subcellular location). Staining concentrated at cut edges or necrotic areas, or retained in a no-primary control, raises concern for processing or detection artefact (standard IHC practice). Endogenous enzyme activity can also produce misleading DAB colour, so verify peroxidase blocking and compare control sections before calling an unexpected cell population positive (standard IHC practice).

Boster reagents

Best RBFOX3 / RNA binding protein fox-1 homolog 3 IHC Antibodies

The IHC-validated anti-RBFOX3 antibody has paraffin-section images from mouse and rat brain and cerebellum (M11954 IHC captions); human reactivity and IF/ICC applications are listed (M11954 catalog).

Real IHC data

Anti-NeuN RBFOX3 Rabbit Monoclonal Antibody

Cat # M11954

View product →

M11954 has IHC images from paraffin-embedded mouse and rat brain and cerebellum sections (M11954 IHC captions). Human, mouse and rat reactivity and IF/ICC applications are listed, but no IF image is supplied (M11954 catalog).

Which to pick: Choose M11954 for tissue IHC: its rabbit monoclonal antibody has paraffin-section images using EDTA retrieval at pH 8.0, 1:50 primary dilution and DAB detection; the fixative is unreported (M11954 catalog; M11954 IHC captions). For IF/ICC, M11954 lists those applications and a 1:50 IF dilution, but the payload supplies no IF image (M11954 catalog). For cross-species work, M11954 lists human, mouse and rat reactivity; its IHC images document mouse and rat sections only (M11954 catalog; M11954 IHC captions).

Each figure is that product's own IHC / IF validation image from its datasheet.

References

UniProt Consortium. UniProt entry A6NFN3 (RFOX3_HUMAN, RNA binding protein fox-1 homolog 3).
Human Protein Atlas. RBFOX3 tissue IHC expression (reliability: Enhanced).
Human Protein Atlas. RBFOX3 subcellular location (ICC-IF): Localized to the nucleoplasm..
Human Protein Atlas. RBFOX3 antibody validation summary (3 antibodies).
Chronic HIV-1 Tat and HIV reduce Rbfox3/NeuN: evidence for sex-related effects. Current HIV research 2015 — PMC4665621.
Rbfox3 Promotes Transformation of MDSC-Like Tumor Cells to Shape Immunosuppressive Microenvironment. Advanced science (Weinheim, Baden-Wurttemberg, Germany) 2025 — PMC11848546.
Hypothermic Protection in Neocortex Is Topographic and Laminar, Seizure Unmitigating, and Partially Rescues Neurons Depleted of RNA Splicing Protein Rbfox3/NeuN in Neonatal Hypoxic-Ischemic Male Piglets. Cells 2023 — PMC10605428.
Naringin ameliorates motor dysfunction and exerts neuroprotective role against vanadium-induced neurotoxicity. AIMS neuroscience 2022 — PMC9826750.
PubMed PMID:14702039 — UniProt-cited evidence.
PubMed PMID:16625196 — UniProt-cited evidence.
PubMed PMID:24215932 — UniProt-cited evidence.