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STX4 / Syntaxin-4 · IHC design guide
Design Immunohistochemistry for STX4
Plan STX4 staining in paraffin sections using the catalog antibody at 2–5 μg/ml (datasheet A05345-1). Compare cytoplasmic and membranous staining with the reported tissue pattern (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
STX4 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 | Cytoplasmic and membranous tissue staining (HPA tissue IHC) | |
| Staining pattern | Glandular cells show cytoplasmic and membranous staining (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A05345-1) | |
| Positive control | Colon+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 conditions consistent across sections. (standard IHC practice; not target-specific) | |
| Caveat | Staining has low consistency with tissue RNA levels (HPA tissue IHC) | |
| Regulation | Rises with neutrophil differentiation (UniProt) | |
| Isoform / epitope | 2 isoforms; map the epitope to cytoplasmic or external sequence (UniProt) |
Section 1
Recommended STX4 IHC & IF Protocols
The catalog antibody’s IHC-P protocol is accompanied by three published STX4 staining protocols for ovarian cancer and clinical specimens (PMC8798739; PMC12144715; PMC10788513).
| Sample | Paraffin-embedded human colon cancer tissue; fixative not specified (datasheet A05345-1) |
| Fixation | Image fixative and duration unreported (datasheet A05345-1); 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 A05345-1); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A05345-1) |
| Primary antibody | Rabbit anti-STX4, 2-5 μg/ml (datasheet A05345-1) |
| Primary incubation | Overnight at 4 °C (datasheet A05345-1) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet A05345-1) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | STX4-positive staining in glandular cells of colon (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic and membranous expression in several tissues. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA pH 8.0 retrieval (datasheet: A05345-1); the ovarian cancer protocols used pressure-cooker EDTA-citrate retrieval (PMC8798739; PMC12144715).
Section 2
What Is the Expected STX4 Staining Pattern?
STX4 should appear chiefly at the cell membrane, with cytoplasmic staining also reported in tissue sections (UniProt Q12846 localization; HPA tissue IHC). Strong examples include gastrointestinal glandular cells, kidney tubular cells, and lymph-node germinal-center cells (HPA tissue IHC: High). HPA rates the tissue pattern Approved while noting low consistency between antibody staining and RNA expression; interpret intensity alongside morphology and controls (HPA tissue IHC).
| Membranous staining, with some cytoplasmic signal, in glandular epithelium or renal tubules. | This fits the reported tissue pattern and the membrane location of STX4 (HPA tissue IHC; UniProt Q12846 localization). Score the identified cell population and compartment together; a brown field alone does not establish a positive result (general IHC practice). |
| Strong staining is confined to nuclei, with little membrane or cytoplasmic signal. | An exclusively nuclear pattern conflicts with the reported membrane and cytoplasmic locations (HPA tissue IHC; UniProt Q12846 localization). Treat it as suspect and check morphology, detection background, and a known-positive section before interpreting it as STX4 (general IHC practice). |
| Adipocytes or cardiomyocytes stain strongly while expected positive cells do not. | HPA reports STX4 as Not detected in adipocytes and cardiomyocytes under its IHC conditions (HPA tissue IHC). Consider cross-reactivity or endogenous detection activity, then compare a reagent control and a known-positive tissue; one unexpected stain does not establish expression (general IHC practice). |
| Pale, widespread color obscures cell borders across positive and comparison tissue. | This is difficult to reconcile with a cell-associated membranous and cytoplasmic pattern (HPA tissue IHC). Check blocking, washing, chromogen development, and detection controls before scoring; diffuse background can make weak cells appear positive (general IHC practice). |
| No convincing signal appears in glandular cells of colon or in kidney tubules. | These are High-staining populations in HPA tissue IHC, so a blank run warrants a technical check (HPA tissue IHC: High). Review section quality, retrieval, primary-antibody conditions, and detection with controls; HPA's low staining–RNA consistency cautions against treating one negative slide as proof of absence (HPA tissue IHC; general IHC practice). |
💡Expected STX4 appearanceCall a section positive when identifiable gastrointestinal glandular cells, kidney tubular cells, or germinal-center cells show clear membranous and/or cytoplasmic staining, potentially strong in those HPA High populations; isolated nuclear color or uniform field-wide haze is suspect (HPA tissue IHC; UniProt Q12846 localization; general IHC practice).
| Membrane topology and compartment | STX4 has a transmembrane segment at residues 276–296 and a cytoplasmic region at 1–275 (UniProt Q12846 topology). A membrane-associated pattern is therefore biologically plausible. The antibody epitope is not given, so topology cannot identify which antigen-retrieval condition will work. |
| Tissue and cell selection | Colon, duodenum, gallbladder, rectum, small intestine, and stomach glandular cells; kidney tubules; and lymph-node germinal-center cells are reported High (HPA tissue IHC). Select and score the named cells, since neighboring cells within a section need not share that reported level (HPA tissue IHC). |
| Strength of the tissue evidence | HPA calls tissue IHC Approved but reports low consistency between staining and RNA expression; antibody HPA001330 is IHC Approved (HPA tissue IHC; HPA antibody validation). Use the observed pattern as a guide, and resolve unexpected staining with controls rather than treating an HPA level as a universal cutoff (general IHC practice). |
| Processing and isoforms | UniProt lists a full-length 1–297 chain, no signal peptide or propeptide, and 2 isoforms (UniProt Q12846 processing; isoforms). No antibody epitope or isoform-specific staining evidence is supplied, so these annotations cannot predict separate IHC patterns or justify a shedding explanation. |
| IF/ICC Q: What localization should a separate IF/ICC assay show? | A: Predominant plasma-membrane signal; HPA approves that subcellular location and rates antibody HPA001330 ICC Enhanced (HPA subcellular ICC-IF; HPA antibody validation). UniProt also lists perinuclear localization and neurite tips in neuronal cells (UniProt Q12846 localization). These observations do not specify an IF/ICC protocol. |
| Situation | Likely cause | Next action |
|---|---|---|
| Positive tissue is blank. | A technical failure is possible when HPA High glandular or tubular cells lack signal (HPA tissue IHC; general IHC practice). | Confirm the named cells are present, then check the run's retrieval, primary-antibody conditions, and detection controls (general IHC practice). No STX4-specific retrieval setting or dilution is supplied. |
| Only nuclei are colored. | This disagrees with reported membrane and cytoplasmic localization (HPA tissue IHC; UniProt Q12846 localization). | Compare with a known-positive section and a detection control; reassess nonspecific color and cell identification before assigning STX4 positivity (general IHC practice). |
| The whole section has diffuse brown haze. | Background can obscure the cell-associated pattern expected for STX4 (HPA tissue IHC; general IHC practice). | Review blocking, washes, and chromogen exposure; use an appropriate negative reagent control to locate background in the detection workflow (general IHC practice). |
| Comparison cells stain as strongly as expected positive cells. | HPA reports adipocytes and cardiomyocytes as Not detected, while several glandular and tubular populations are High (HPA tissue IHC). | Verify cell identity and compare controlled sections. Investigate nonspecific antibody binding or endogenous detection activity; do not infer a new tissue distribution from one section (general IHC practice). |
| A positive cell population shows cytoplasmic color without a crisp surface rim. | HPA describes tissue staining as cytoplasmic and membranous, so cytoplasmic color alone is not automatically incompatible (HPA tissue IHC). | Judge whether staining remains cell-associated and occurs in the expected cells; compare a controlled positive section before rejecting or accepting the result (HPA tissue IHC; general IHC practice). |
| An HPA Not detected tissue has faint staining. | Not detected describes the HPA observation under its conditions, not a proven universal absence of STX4 (HPA tissue IHC). | Score intensity and cell type against the same run's positive and reagent controls; report the observation cautiously if it persists (general IHC practice). |
Experiment evidence
IHC & IF images showing STX4 staining
A05345-1 · IHCIHC analysis of Syntaxin 4/STX4 using anti-Syntaxin 4/STX4 antibody (A05345-1). Syntaxin 4/STX4 was detected in a paraffin-embedded section
A05345-1 · IHCIHC analysis of Syntaxin 4/STX4 using anti-Syntaxin 4/STX4 antibody (A05345-1). Syntaxin 4/STX4 was detected in a paraffin-embedded section
A05345-1 · IFIF analysis of Syntaxin 4/STX4 using anti-Syntaxin 4/STX4 antibody (A05345-1). Syntaxin 4/STX4 was detected in an immunocytochemical section
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000103496-STX4/tissue/cerebral+cortex#imid_513410
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000103496-STX4/tissue/colon#imid_513344
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000103496-STX4/tissue/liver#imid_513337
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000103496-STX4/tissue/kidney#imid_513338
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA001330; green = STX4, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA001330; green = STX4, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for STX4 IHC & IF
🧪Run colon first: glandular cells should stain (HPA: High in colon glandular cells). Use adipose tissue adipocytes as the negative tissue (HPA: Not detected in adipocytes); on the colon slide, assess adjacent nonglandular cells for background staining, without assuming they are STX4-negative (HPA: colon result specifies glandular cells only).
Positive control tissue: Colon (Glandular cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show STX4 in A-431, U-251MG, U2OS, U2OS, siRNA 2 (10x), U2OS, scrambled (10x), with annotated localisation: Plasma membrane (approved) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control; a host-matched rabbit IgG control matched to the primary antibody’s clonality; and a validated STX4 knockout specimen or peptide-block control where the immunizing peptide is available (caption: rabbit primary; standard IHC practice). Quench endogenous peroxidase and inspect glandular lumens for trapped chromogen on the colon section (standard IHC practice).
⚠️Feasibility: A target-specific fixation window and fixation effect are unreported; the selected A05345-1 paraffin-section caption does not state the fixative (selected-SKU tissue-IHC caption). The reported IHC example uses heat retrieval in EDTA at pH 8.0, so test retrieval under that condition, but its necessity is unreported (selected-SKU tissue-IHC caption). The supplied evidence does not establish whether frozen sections or IF are easier; HPA does show approved plasma-membrane ICC-IF localization, while luminal chromogen trapping is a potential colon-section artefact (HPA: subcellular; standard IHC practice).
HPA tissue IHC evidence for STX4
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Low 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 |
|---|---|---|---|---|
| Colon | Glandular cells | High | Protein (IHC) | HPA → |
| Duodenum | Glandular cells | High | Protein (IHC) | HPA → |
| Gallbladder | Glandular cells | High | Protein (IHC) | HPA → |
| Kidney | Cells in tubules | High | Protein (IHC) | HPA → |
| Lymph node | Germinal center cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Caudate | Glial cells | Not detected | Protein (IHC) | HPA → |
| Heart muscle | Cardiomyocytes | Not detected | Protein (IHC) | HPA → |
| Hippocampus | Glial cells | Not detected | Protein (IHC) | HPA → |
| Soft tissue | Fibroblasts | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced STX4 IHC Tips
Troubleshoot STX4 staining in paraffin sections by checking retrieval, signal location, background and scoring against the documented IHC conditions.
Which retrieval conditions should I try first for weak STX4 staining in paraffin sections?
Start with heat-mediated antigen retrieval in EDTA at pH 8.0 for paraffin sections (datasheet A05345-1). The catalog antibody produced chromogenic staining after this retrieval, followed by 2 μg/ml primary antibody overnight at 4°C (datasheet A05345-1). If staining is weak, compare controlled changes in heating duration on adjacent sections while keeping the antibody concentration and DAB development consistent (standard IHC practice). Assess whether expected membranous and cytoplasmic staining improves without a parallel rise in diffuse tissue background; those locations are supported for STX4, but the caption does not establish an optimal heating duration (HPA tissue IHC; datasheet A05345-1).
How should I investigate inconsistent STX4 staining between paraffin section batches?
Target-specific fixation sensitivity is unknown: the selected tissue-IHC caption describes a paraffin-embedded section but does not state its fixative (datasheet A05345-1). Record each batch’s fixative, fixation duration, processing history and section age, then compare batches using the same EDTA pH 8.0 retrieval and staining run (standard IHC practice; datasheet A05345-1). Include a previously stained control section and hold the primary antibody at 2 μg/ml overnight at 4°C while investigating the difference (datasheet A05345-1). Interpret a batch-wide loss of signal as a processing or staining problem until matched controls distinguish it from tissue variation (standard IHC practice).
What staining pattern is credible for STX4 in chromogenic IHC?
Evaluate membrane-associated staining first, while allowing cytoplasmic signal: tissue IHC reports both patterns, and plasma membrane localisation is approved in subcellular imaging (HPA tissue IHC; HPA subcellular). STX4 has a single transmembrane segment at residues 276–296, with residues 1–275 on the cytoplasmic side (UniProt Q12846 topology). Perinuclear localisation is also recorded, so a restricted perinuclear component can be assessed alongside the membrane pattern rather than scored alone (UniProt Q12846 subcellular). Compare signal within intact cells against neighbouring negative areas and the expected compartment; uniform nuclear colour or staining confined to damaged edges warrants investigation (standard IHC practice).
Can this IHC stain distinguish STX4 isoforms or an extracellular epitope?
Do not assign isoform-specific staining from this image: STX4 has 2 recorded isoforms, while the supplied catalog caption does not map the antibody epitope (UniProt Q12846 isoforms; datasheet A05345-1). The canonical protein places residues 1–275 cytoplasmically, residues 276–296 across the membrane and residue 297 extracellularly (UniProt Q12846 topology). Phosphorylation is recorded at several residues, including serines 14 and 117, but its effect on this antibody’s IHC signal is unknown (UniProt Q12846 modified residues; datasheet A05345-1). For an isoform or epitope claim, obtain the antibody’s mapped immunogen and compare appropriate orthogonal controls under the same retrieval conditions (standard IHC practice).
How should I adapt the STX4 question to multiplex immunofluorescence?
For multiplex IF, pair STX4 with a marker identifying the expected cell population, such as glandular cells in colon, and assess membrane signal within that population (HPA tissue IHC; HPA subcellular). Choose spectrally separated fluorophores and consider a longer-wavelength channel for STX4 when tissue autofluorescence compromises shorter-wavelength channels; include single-stain and unstained controls (standard IF practice). Because the supplied catalog caption does not identify the antibody epitope, compare permeabilised staining for a cytoplasmic epitope with nonpermeabilised surface staining only if extracellular epitope binding is established (datasheet A05345-1; UniProt Q12846 topology; standard IF practice). Treat this as IF assay development: the selected-SKU evidence documents paraffin-section chromogenic IHC, while HPA subcellular images do not establish IF performance for that SKU (datasheet A05345-1; HPA subcellular).
What should I check when STX4 DAB staining looks diffuse?
First compare the stained section with a no-primary control and inspect whether colour follows intact cell boundaries or appears broadly in damaged tissue (standard IHC practice). The documented run used 10% goat serum blocking, 2 μg/ml primary antibody overnight at 4°C, a peroxidase-conjugated secondary for 30 minutes at 37°C, and DAB detection (datasheet A05345-1). Check the peroxidase block, washing and DAB development as general chromogenic workflow variables, rather than treating them as STX4-specific evidence (standard IHC practice). If background rises with stronger retrieval or longer development, compare adjacent sections under matched detection settings before changing the interpretation of weak membranous signal (standard IHC practice; HPA subcellular).
How can I quantify STX4 staining without conflating expression and tissue composition? ⚠ ANSWER MARKED FOR VERIFICATION
Define the scored population and compartment before reading slides; for colon sections, glandular cells provide a documented STX4-positive population (HPA tissue IHC). An H-score combines the percentage of cells at intensity grades 0–3 into a 0–300 score, while percentage positive cells or positive area per mm² can answer different questions (standard IHC practice). Normalise cell-based measures to the number of evaluable cells in that population, or area-based measures to viable tissue area, and exclude necrotic or folded regions (standard IHC practice). Keep retrieval, DAB development, thresholds and compartment rules fixed across groups; HPA reports low consistency between antibody staining and RNA expression, so avoid treating RNA as a direct calibration (standard IHC practice; HPA tissue IHC).
How do I distinguish credible STX4 signal from IHC artefact?
A credible result places staining at the membrane, potentially with cytoplasmic or perinuclear signal, in intact cells rather than relying on diffuse colour alone (HPA tissue IHC; HPA subcellular; UniProt Q12846 subcellular). Compare with documented positive populations such as colon glandular cells or kidney tubule cells and check whether staining remains anatomically coherent across the section (HPA tissue IHC; standard IHC practice). Treat isolated nuclear colour, strong section-edge staining, necrotic deposits and signal in a no-primary control as prompts to inspect detection and tissue quality (standard IHC practice). Check endogenous peroxidase and DAB development when colour appears outside plausible compartments, and weigh apparent negatives cautiously because HPA labels its tissue staining approved while reporting low consistency with RNA expression (standard IHC practice; HPA tissue IHC).
Boster reagents
Best STX4 / Syntaxin-4 IHC Antibodies
Anti-STX4 antibody A05345-1 has IHC images from human colon cancer and rat brain paraffin sections, plus IF data from A549 cells (catalog image captions). Human, mouse and rat reactivity is listed (catalog).
Real IHC data
Anti-Syntaxin 4/STX4 Antibody ®
Cat # A05345-1
A05345-1 will render with its IHC figure from a human colon cancer paraffin section; the catalog also shows IHC in a rat brain paraffin section (catalog IHC captions). The same SKU is listed for IHC and IF/ICC and has an IF image from A549 cells (catalog applications and IF caption).
Which to pick: Choose A05345-1 for paraffin-section IHC: its human colon cancer and rat brain captions document EDTA retrieval at pH 8.0 and 2 μg/ml primary antibody (catalog IHC captions); the fixative is unreported. Choose the same SKU for IF/ICC in A549 cells, supported by its 5 μg/ml IF caption and IF/ICC application listing (catalog). For cross-species planning, human, mouse and rat reactivity is listed, while the supplied IHC images document human and rat samples; the antibody is rabbit-hosted, and clonality is unreported (catalog reactivity, host and IHC captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q12846 (STX4_HUMAN, Syntaxin-4).
- Human Protein Atlas. STX4 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. STX4 subcellular location (ICC-IF): Localized to the plasma membrane..
- Human Protein Atlas. STX4 antibody validation summary (1 antibodies).
- STX4 expression is associated with classification, clinical stage and lymphatic metastasis in ovarian cancer. Translational cancer research 2019 — PMC8798739.
- Silencing of STX4 inhibits the proliferation, migration and invasion of ovarian cancer cells via EMT/MMP2/ CCND1 signaling pathway. Journal of ovarian research 2025 — PMC12144715.
- STX4 as a potential biomarker for predicting prognosis and guiding clinical treatment decisions in clear cell renal cell carcinoma. Heliyon 2024 — PMC10788513.
- Syntaxin 4 Expression in Pancreatic β-Cells Promotes Islet Function and Protects Functional β-Cell Mass. Diabetes 2018 — PMC6245223.
- PubMed PMID:8206394 — UniProt-cited evidence.
- PubMed PMID:8760387 — UniProt-cited evidence.
- PubMed PMID:10080545 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for STX4 (Syntaxin-4). Expected localisation Cytoplasmic and membranous tissue staining (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A05345-1). Validated IHC antibody: A05345-1 at 2-5 μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Colon (Glandular cells); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human colon cancer tissue; fixative not specified (datasheet A05345-1); 2. Fixation: Image fixative and duration unreported (datasheet A05345-1); 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 A05345-1); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A05345-1); 8. Primary antibody: Rabbit anti-STX4, 2-5 μg/ml (datasheet A05345-1); 9. Primary incubation: Overnight at 4 °C (datasheet A05345-1). Watch for: Fixation: Keep fixation conditions consistent across sections. (standard IHC practice; not target-specific); Caveat: Staining has low consistency with tissue RNA levels (HPA tissue IHC); Isoform / epitope: 2 isoforms; map the epitope to cytoplasmic or external sequence (UniProt); Regulation: Rises with neutrophil differentiation (UniProt). Evidence: UniProt Q12846, HPA tissue IHC · Approved, HPA ICC-IF · plasma membrane., Datasheet A05345-1, PMC8798739, PMC12144715. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/STX4/immunohistochemistry-ihc-guide.