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SEPTIN3 / Neuronal-specific septin-3 · IHC design guide
Design Immunohistochemistry for SEPTIN3
Plan paraffin-section SEPTIN3 IHC around high cerebral cortex neuropil staining (HPA tissue IHC). Compare with an HPA-negative tissue and use the catalog antibody’s chromogenic IHC workflow (HPA tissue IHC; datasheet A30742-1).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
SEPTIN3 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 | Cerebral cortex neuropil staining (HPA tissue IHC); cytoplasmic and synaptic location expected (UniProt) | |
| Staining pattern | High staining in cerebral cortex neuropil (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A30742-1) | |
| Positive control | Cerebral cortex+2 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 sections (standard IHC practice; not target-specific); Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A30742-1) | |
| Caveat | Presumed off-target staining may complicate interpretation (HPA tissue IHC) | |
| Regulation | Brain-specific expression (UniProt) | |
| Isoform / epitope | 3 isoforms; antibody epitope coverage is unspecified (UniProt) |
Section 1
Recommended SEPTIN3 IHC & IF Protocols
The catalog antibody’s IHC-P protocol uses heat-mediated EDTA retrieval (datasheet A30742-1). One published SEPT3 IHC protocol provides a citrate-buffer comparison in TNBC tissue (PMC11057513).
| Sample | Paraffin-embedded mouse brain tissue; fixative not specified (datasheet A30742-1) |
| Fixation | Image fixative and duration unreported (datasheet A30742-1); 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 A30742-1); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A30742-1) |
| Primary antibody | Rabbit anti-SEPTIN3, 2-5 μg/ml (datasheet A30742-1) |
| Primary incubation | Overnight at 4 °C (datasheet A30742-1) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet A30742-1) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | SEPTIN3-positive staining in neuropil of cerebral cortex (HPA tissue IHC: High). HPA tissue profile: Selective expression in the CNS. No signal in the no-primary control. |
💡Decision noteStart with EDTA pH 8.0 heat retrieval (datasheet A30742-1); consider citrate buffer during optimization (PMC11057513).
Section 2
What Is the Expected SEPTIN3 Staining Pattern?
SEPTIN3 is a cytoplasmic, cytoskeletal and synaptic protein with no transmembrane segment (UniProt Q9UH03). In paraffin sections, expect strong staining in cerebral cortex neuropil (HPA: High); HPA also reports high Sertoli cell and medium pancreatic endocrine cell staining (HPA: tissue IHC). Interpret those non-CNS signals cautiously: HPA describes CNS-selective expression, rates its tissue profile Supported, notes presumed off-target binding and awaits external verification (HPA: tissue IHC).
| Strong staining in cerebral cortex neuropil, concentrated in the neuropil rather than neuronal nuclei (HPA: High in cortex neuropil; UniProt Q9UH03 localization). | This matches the clearest supplied positive tissue pattern. Assess the neuropil separately from cell bodies and background; a positive control should reproduce the expected location as well as the staining intensity (HPA: tissue IHC; general IHC practice). |
| Prominent nuclear staining with little cortical neuropil staining (HPA: High in cortex neuropil). | The compartment disagrees with the principal tissue pattern and UniProt's cytoplasmic, cytoskeletal and synaptic annotation (HPA: tissue IHC; UniProt Q9UH03 localization). Check detection background and antibody specificity before interpreting nuclear signal as SEPTIN3 (general IHC practice). |
| Strong staining in an HPA-listed negative cell population, such as adipocytes in adipose tissue (HPA: Not detected in adipocytes). | Treat this as discordant with the reference pattern. Cross-reactivity or endogenous detection activity is possible; review the no-primary control and the location of staining before assigning target expression (HPA: tissue IHC; general IHC practice). |
| Diffuse color across tissue compartments and empty areas of the slide (general IHC practice). | A pattern that does not track the expected neuropil is difficult to score as specific SEPTIN3 staining (HPA: High in cortex neuropil). Review blocking, washes, chromogen development and the no-primary control as general IHC checks, without assuming a SEPTIN3-specific cause (general IHC practice). |
| No detectable staining in cerebral cortex neuropil (HPA: High in cortex neuropil). | The run has failed to reproduce the strongest supplied tissue reference. First verify that the positive tissue and detection controls worked; then review the validated IHC-P workflow, including retrieval and antibody use, without assuming SEPTIN3 has a known fixation sensitivity (HPA: tissue IHC; general IHC practice). |
💡Expected SEPTIN3 appearanceCall a result consistent when cerebral cortex neuropil shows strong, localized staining (HPA: High in cortex neuropil; UniProt Q9UH03 localization); isolated nuclear signal or strong signal in HPA-listed negative cells warrants specificity checks (HPA: tissue IHC; general IHC practice).
| Tissue and cell context | Cerebral cortex neuropil is the strongest supplied CNS reference (HPA: High). High Sertoli cell and medium pancreatic endocrine cell staining are reported, but HPA also describes CNS selectivity and presumed off-target binding; use these non-CNS results as observations requiring caution (HPA: tissue IHC). |
| Compartment and topology | UniProt places SEPTIN3 in the cytoplasm, cytoskeleton and synapse and lists no transmembrane segment (UniProt Q9UH03). This supports assessing intracellular and neuropil patterns; it does not establish a membrane-only IHC pattern (UniProt Q9UH03; HPA: tissue IHC). |
| Antibody evidence | Two listed antibodies have Supported IHC status, while HPA's overall tissue profile is Supported and pending external verification (HPA: HPA003548, CAB017633; HPA: tissue IHC). A matching pattern strengthens interpretation but does not by itself resolve the reported off-target concern (HPA: tissue IHC). |
| Isoforms and target-specific retrieval | UniProt lists three isoforms, but the supplied record gives no antibody epitope or isoform-specific IHC staining data (UniProt Q9UH03; HPA: antibody summary). Target-specific fixation sensitivity and retrieval effects are unreported here; use the selected antibody's validated IHC-P instructions for the run (general IHC practice). |
| IF/ICC Q&A: what location can be expected? | HPA reports actin filaments and primary cilium as approved locations, with plasma membrane and nucleoplasm marked uncertain (HPA: subcellular ICC-IF). Those ICC-IF observations should be interpreted in their own application and do not replace the cortical neuropil reference for IHC (HPA: subcellular ICC-IF; HPA: tissue IHC). |
| Situation | Likely cause | Next action |
|---|---|---|
| Cortex neuropil is blank despite a completed IHC run (HPA: High in cortex neuropil). | The result conflicts with the positive tissue reference; a failed detection step or unsuitable assay conditions are possible (HPA: tissue IHC; general IHC practice). | Check the positive and no-primary controls, reagent activity and the selected antibody's IHC-P instructions before scoring the sample negative (general IHC practice). |
| Nuclei dominate the stain while cortex neuropil remains weak (HPA: High in cortex neuropil). | The dominant compartment conflicts with UniProt's cytoplasmic, cytoskeletal and synaptic annotation (UniProt Q9UH03). | Compare the no-primary control, inspect where stain accumulates and review antibody specificity; avoid calling nuclear signal a validated IHC pattern from these sources (general IHC practice; HPA: tissue IHC). |
| Strong staining appears in adipocytes from adipose tissue (HPA: Not detected in adipocytes). | This is discordant with the supplied negative reference; off-target binding or endogenous detection activity may contribute (HPA: tissue IHC; general IHC practice). | Compare negative and positive reference tissues in the same run, then inspect the no-primary control and detection chemistry (general IHC practice). |
| Sertoli cells stain strongly, but the result seems at odds with brain enrichment (HPA: High in Sertoli cells; HPA: RNA tissue enriched in brain). | HPA reports this IHC observation while also noting CNS selectivity and presumed off-target binding (HPA: tissue IHC). | Record the observed cell-specific pattern and its caveat; do not treat Sertoli cell staining alone as proof of SEPTIN3 specificity (HPA: tissue IHC). |
| Brown signal is widespread or present in areas without the expected cortical neuropil pattern (HPA: High in cortex neuropil). | General IHC background or endogenous detection activity can obscure a localized result (general IHC practice). | Inspect the no-primary control and review blocking, washing and development; score target signal only where the tissue and compartment pattern supports it (general IHC practice; HPA: tissue IHC). |
| A result changes after retrieval or processing adjustments (general IHC practice). | These sources provide no SEPTIN3-specific fixation or retrieval effect, so the change cannot be assigned a target-specific mechanism (UniProt Q9UH03; HPA: tissue IHC). | Compare runs using the same positive reference and controls, follow the chosen IHC-P instructions and document the changed condition (general IHC practice). |
Experiment evidence
IHC & IF images showing SEPTIN3 staining
A30742-1 · IHCIHC analysis of Septin 3/SEPTIN3 using anti-Septin 3/SEPTIN3 antibody (A30742-1). Septin 3/SEPTIN3 was detected in a paraffin-embedded secti
A30742-1 · IHCIHC analysis of Septin 3/SEPTIN3 using anti-Septin 3/SEPTIN3 antibody (A30742-1). Septin 3/SEPTIN3 was detected in a paraffin-embedded secti
A30742-1 · IFIF analysis of Septin 3/SEPTIN3 using anti-Septin 3/SEPTIN3 antibody (A30742-1). Septin 3/SEPTIN3 was detected in an immunocytochemical sect
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000100167-SEPTIN3/tissue/cerebral+cortex#imid_20921159
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000100167-SEPTIN3/tissue/colon#imid_20921077
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000100167-SEPTIN3/tissue/liver#imid_20921078
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000100167-SEPTIN3/tissue/kidney#imid_20921071
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA003548; green = SEPTIN3, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA003548; green = SEPTIN3, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for SEPTIN3 IHC & IF
🧪Run cerebral cortex first and look for staining in the neuropil (HPA: High in cerebral cortex neuropil). Use adipose tissue as a negative comparator (HPA: Not detected in adipocytes); the supplied cortex row identifies no internal negative cell population, so assess any unstained cells against the no-primary background (HPA: cortex neuropil row; standard IHC practice).
Positive control tissue: Cerebral cortex (Neuropil, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show SEPTIN3 in A-431, U-251MG, U2OS, ASC52telo, RPTEC/TERT1, hTERT-RPE1 (serum starved), with annotated localisation: Plasma membrane (uncertain), Actin filaments (approved) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only section, a host- and clonality-matched isotype control, and a SEPTIN3 knockout section if available (standard IHC practice; caption: rabbit primary antibody). For HRP/DAB staining of brain, quench endogenous peroxidase and check the no-primary section for tissue background (caption: HRP/DAB detection; standard IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the exact A30742-1 mouse brain caption does not state the fixative (caption: fixative unreported). Heat retrieval in EDTA at pH 8.0 was used for the pictured paraffin section, but a retrieval requirement has not been established (caption: EDTA pH 8.0). The supplied evidence does not establish that frozen sections or IF are easier; in cortex, dense neuropil staining warrants careful comparison with background (HPA: High in cerebral cortex neuropil; standard IHC practice).
HPA tissue IHC evidence for SEPTIN3
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Supported — High consistency between antibody staining and RNA expression data. Presumed off target binding observed and disregarded. 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 |
|---|---|---|---|---|
| Cerebral cortex | Neuropil | High | Protein (IHC) | HPA → |
| Testis | Sertoli cells | High | Protein (IHC) | HPA → |
| Pancreas | Pancreatic endocrine 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 | Glandular 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 SEPTIN3 IHC Tips
Troubleshoot SEPTIN3 staining in paraffin sections by checking retrieval, tissue morphology, cellular distribution, and controls against the reported brain staining pattern (datasheet A30742-1; HPA tissue IHC).
How should I retrieve SEPTIN3 in paraffin sections when brain staining is weak?
Start with heat mediated retrieval in EDTA at pH 8.0 for paraffin SEPTIN3 sections (datasheet A30742-1). This condition accompanies mouse brain staining with 2 μg/ml primary antibody overnight at 4°C; keep the antibody and detection conditions constant while assessing retrieval (datasheet A30742-1; standard IHC practice). Examine matched sections for neuropil signal and preserved morphology, since excessive heating can damage sections and complicate interpretation (HPA: cerebral cortex neuropil High; standard IHC practice). If staining remains weak, compare another buffered retrieval condition as a fallback, changing one variable at a time and recording the heat cycle (standard IHC practice).
Could fixation explain inconsistent SEPTIN3 staining across paraffin brain samples?
The selected paraffin mouse brain caption does not state a fixative, so target specific fixation sensitivity for SEPTIN3 is unknown (datasheet A30742-1). Record each sample’s fixative and fixation duration, then compare similarly processed sections under the same EDTA pH 8.0 retrieval and detection conditions (datasheet A30742-1; standard IHC practice). Include a reference brain section in each run and assess both neuropil staining and tissue preservation before attributing a difference to fixation (HPA: cerebral cortex neuropil High; standard IHC practice). Do not infer fixation tolerance from SEPTIN3’s cytoplasmic location or its 91 phosphoserine annotation; neither establishes a fixation response (UniProt Q9UH03; standard IHC practice).
What staining pattern should I expect, and how should I assess unexpected compartments?
Assess cerebral cortex neuropil as a useful positive pattern, while expecting SEPTIN3 association with cytoplasm, cytoskeleton, and synapses (HPA: cerebral cortex neuropil High; UniProt Q9UH03). Septins can form filaments and associate with actin filaments, microtubules, and cellular membranes, so process associated staining warrants careful morphological review (UniProt Q9UH03). HPA subcellular data approve actin filament and primary cilium locations, but mark plasma membrane and nucleoplasm locations uncertain; those observations do not establish a paraffin tissue pattern (HPA subcellular). For predominantly nuclear or diffuse staining, compare matched negative and positive tissues and review antibody controls before assigning SEPTIN3 localisation (HPA tissue IHC; standard IHC practice).
Can this IHC stain distinguish SEPTIN3 isoforms or a phosphorylation dependent epitope?
SEPTIN3 has 3 annotated isoforms, but the supplied antibody caption does not identify its epitope or establish isoform selectivity (UniProt Q9UH03; datasheet A30742-1). The septin type G domain spans residues 58–331, and phosphoserine is annotated at residue 91; neither fact shows what this antibody recognizes (UniProt Q9UH03). Treat chromogenic signal as antibody reactivity until specificity is supported with an appropriate loss of target control or independently validated reagent (standard IHC practice). If a biological comparison depends on one isoform or phosphorylation state, obtain direct epitope validation before assigning that interpretation to differences in staining (standard IHC practice).
How can I evaluate SEPTIN3 by IF without overinterpreting the paraffin IHC result?
For exploratory IF or ICC, multiplex SEPTIN3 with a validated neuronal marker to assess the expected cell population and inspect signal around neuronal processes (UniProt Q9UH03: brain specific, synapse; standard IF practice). Choose spectrally separated fluorophores and an emission channel with low sample autofluorescence, checking single stain and no primary controls before interpreting overlap (standard IF practice). SEPTIN3 has no transmembrane segment and is annotated in cytoplasm and cytoskeleton, so titrate permeabilisation for intracellular epitope access while checking that cellular structure remains intact (UniProt Q9UH03; standard IF practice). The 2 μg/ml overnight condition documents paraffin chromogenic IHC and does not establish an IF dilution (datasheet A30742-1).
How do I separate SEPTIN3 signal from chromogenic background?
Start by comparing no primary and secondary only controls with the stained section, and inspect whether DAB deposits follow tissue edges, debris, or expected neuropil (standard IHC practice; HPA: cerebral cortex neuropil High). The selected mouse brain example used 10% goat serum blocking, 2 μg/ml primary antibody overnight at 4°C, and peroxidase based DAB detection (datasheet A30742-1). Include an endogenous peroxidase block and optimize washing and antibody concentration if control sections develop color; these are general chromogenic IHC measures (standard IHC practice). Do not assume every nonbrain signal is background, because HPA reports high staining in Sertoli cells and medium staining in pancreatic endocrine cells (HPA tissue IHC).
How should I quantify SEPTIN3 when much of the signal lies in neuropil? ⚠ ANSWER MARKED FOR VERIFICATION
Define a consistent anatomical region and DAB threshold before scoring, then measure positive area or optical density per mm² of evaluable neuropil (HPA: cerebral cortex neuropil High; standard IHC practice). Normalize the measurement to neuropil area, and account for tissue loss, section quality, and differences in neuronal content across samples (standard IHC practice). For a cell based question, report the percentage of positive cells or an H score from 0–300, with explicit cell identification and intensity categories (standard IHC practice). Keep neuropil and cell scores separate, and use matched staining runs and blinded review to limit variation from processing and threshold choices (standard IHC practice).
What findings would make a SEPTIN3 positive IHC result convincing?
A convincing result follows reproducible brain neuropil staining and the expected cytoplasmic, cytoskeletal, or synaptic distribution, with appropriate control sections remaining clean (HPA: cerebral cortex neuropil High; UniProt Q9UH03; standard IHC practice). Check cell identity before calling a signal neuronal: HPA also reports staining in Sertoli cells and pancreatic endocrine cells, while its tissue assessment notes presumed off target binding (HPA tissue IHC). Predominantly nucleoplasmic staining needs additional scrutiny because that subcellular assignment is uncertain, although actin filament localisation is approved (HPA subcellular). Exclude edge effects, necrotic areas, and endogenous enzyme deposits using morphology and no primary controls before interpreting an unusual pattern (standard IHC practice).
Boster reagents
Best SEPTIN3 / Neuronal-specific septin-3 IHC Antibodies
A30742-1 has IHC images from human, mouse and rat paraffin brain sections (catalog IHC captions), plus an IF image from SiHa cells (catalog IF caption).
Real IHC data
Anti-Septin 3/SEPTIN3 Antibody ®
Cat # A30742-1
A30742-1 is the sole card and shows SEPTIN3 staining in paraffin brain sections from human, mouse and rat (catalog IHC captions). It is also listed for ICC/IF and has an IF image from SiHa cells (catalog applications; catalog IF caption).
Which to pick: Choose A30742-1 for paraffin-section IHC: its captions document staining in human, mouse and rat brain using 2 μg/ml primary antibody after heat retrieval in EDTA, pH 8.0 (catalog IHC captions). For IF/ICC, A30742-1 is listed for both applications and has a SiHa cell IF image at 5 μg/ml; its listed reactivity spans human, mouse and rat, while clonality is unreported (catalog applications; catalog IF caption; catalog reactivity; catalog clone field). The IHC captions describe paraffin sections but do not report the fixative (catalog IHC captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q9UH03 (SEPT3_HUMAN, Neuronal-specific septin-3).
- Human Protein Atlas. SEPTIN3 tissue IHC expression (reliability: Supported).
- Human Protein Atlas. SEPTIN3 subcellular location (ICC-IF): Mainly localized to the plasma membrane and actin filaments. In addition localized to the nucleoplasm and primary cilium..
- Human Protein Atlas. SEPTIN3 antibody validation summary (2 antibodies).
- SEPT3 as a Potential Molecular Target of Triple-Negative Breast Cancer. International journal of general medicine 2024 — PMC11057513.
- PubMed PMID:11322766 — UniProt-cited evidence.
- PubMed PMID:15461802 — UniProt-cited evidence.
- PubMed PMID:17974005 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for SEPTIN3 (Neuronal-specific septin-3). Expected localisation Cerebral cortex neuropil staining (HPA tissue IHC); cytoplasmic and synaptic location expected (UniProt). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A30742-1). Validated IHC antibody: A30742-1 at 2-5 μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Cerebral cortex (Neuropil); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded mouse brain tissue; fixative not specified (datasheet A30742-1); 2. Fixation: Image fixative and duration unreported (datasheet A30742-1); 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 A30742-1); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A30742-1); 8. Primary antibody: Rabbit anti-SEPTIN3, 2-5 μg/ml (datasheet A30742-1); 9. Primary incubation: Overnight at 4 °C (datasheet A30742-1). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific); Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A30742-1); Caveat: Presumed off-target staining may complicate interpretation (HPA tissue IHC); Isoform / epitope: 3 isoforms; antibody epitope coverage is unspecified (UniProt); Regulation: Brain-specific expression (UniProt). Evidence: UniProt Q9UH03, HPA tissue IHC · Supported, HPA ICC-IF · Mainly localized to the plasma membrane and actin filaments. In…, Datasheet A30742-1, PMC11057513. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/SEPTIN3/immunohistochemistry-ihc-guide.