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VASP / Vasodilator-stimulated phosphoprotein · IHC design guide
Design Immunohistochemistry for VASP
Plan VASP IHC in paraffin sections around the cytoplasmic staining observed in most cells (HPA tissue IHC). Compare staining across cell types, while treating focal-adhesion localisation as a molecular expectation rather than an established tissue pattern (UniProt).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
VASP 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 | Cytoplasm in most cells (HPA tissue IHC) | |
| Staining pattern | Most cells show cytoplasmic staining (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet M00303) | |
| Positive control | Appendix+4 more · see all | |
| Negative control | Heart muscle+1 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image M00303) | |
| Caveat | Widespread staining limits cell-type specificity (HPA tissue IHC) | |
| Regulation | Thrombin/PMA recruit VASP to focal adhesions (UniProt) | |
| Isoform / epitope | No annotated isoforms; chain 2–380 has no extracellular domain (UniProt) |
Section 1
Recommended VASP IHC & IF Protocols
The catalog antibody’s IHC-P protocol is accompanied by published VASP IHC protocols for breast cancer tissue and colorectal cancer tissue (PMC5216765; PMC6419247).
| Sample | Paraffin-embedded human liver cancer tissue; fixative not specified (datasheet M00303) |
| Fixation | Image fixative and duration unreported (datasheet M00303); 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 M00303); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet M00303) |
| Primary antibody | Rabbit monoclonal (clone BHO-22) anti-VASP, 1:50 (datasheet M00303) |
| Primary incubation | Overnight at 4 °C (datasheet M00303) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet M00303) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | VASP-positive staining in glandular cells of appendix (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression in most cells. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA retrieval at pH 8.0 for the catalog antibody (datasheet M00303); the published antibody protocols used different retrieval conditions (PMC5216765; PMC6419247).
Section 2
What Is the Expected VASP Staining Pattern?
VASP is mainly cytoplasmic in tissue sections, with localization also reported at the cytoskeleton, cell junctions and focal adhesions (HPA tissue IHC; UniProt P50552). Expect staining across many cell types, including strong staining in several glandular, respiratory epithelial, endothelial and hematopoietic populations (HPA tissue IHC). Interpret that breadth with care: HPA rates the tissue profile Enhanced but reports only medium consistency with RNA expression (HPA tissue IHC). VASP has no transmembrane segment (UniProt P50552 topology).
| Cytoplasmic staining in appendix or breast glandular cells, bronchial respiratory epithelium, colon endothelium, or bone marrow hematopoietic cells (HPA tissue IHC). | These are reported high-staining cell populations and useful positive references (HPA tissue IHC). Judge the named cells within their tissue context; the HPA profile describes cytoplasmic expression in most cells, so a positive result need not be confined to one population (HPA tissue IHC). |
| Staining appears predominantly nuclear, with little cytoplasmic signal (HPA tissue IHC; UniProt P50552 subcellular location). | That distribution does not match the reported tissue profile or annotated locations (HPA tissue IHC; UniProt P50552 subcellular location). Treat it as suspect and review the detection controls and staining conditions before assigning biological meaning (general IHC practice). |
| Strong staining is assigned to cardiomyocytes or skeletal myocytes (HPA tissue IHC). | HPA reports VASP as not detected in those specific cell types (HPA tissue IHC). Check cell identification and a reagent control; cross-reactivity or endogenous detection activity are possible explanations, but this pattern alone cannot distinguish them (general IHC practice). |
| Color covers extracellular areas, tissue edges, or many compartments without a discernible cellular pattern. | This is background rather than a convincing VASP localization call (general IHC practice). Assess staining without primary antibody and review blocking, washing, chromogen development and section condition before scoring cells (general IHC practice). |
| No cellular signal appears in an otherwise assessable positive-reference section, such as appendix glandular cells (HPA: High). | A failed stain is plausible, but absence alone cannot identify the cause (general IHC practice). Check that the expected cells are present, then review the antibody, retrieval and detection workflow with controls (general IHC practice). |
💡Expected VASP appearanceCall VASP positive when identifiable cells show cytoplasmic staining, especially in an HPA high-staining population (HPA tissue IHC); reject widespread acellular color or isolated dominant nuclear staining as unconvincing localization (general IHC practice; HPA tissue IHC).
| Tissue and cell choice | HPA reports high staining in specified glandular, respiratory epithelial, endothelial and hematopoietic cells, but low staining in glia, fibroblasts and adipocytes (HPA tissue IHC). A weak result in a low-staining population is less informative than one in a high-staining reference (general IHC practice). |
| Topology and processing | VASP has no transmembrane segment or signal peptide, and its annotated chain spans residues 2–380 (UniProt P50552 topology; UniProt P50552 processing). Membrane-adjacent staining can still be credible because VASP localizes to cell projections, junctions and focal adhesions (UniProt P50552 subcellular location). |
| Modification and antibody scope | VASP has annotated phosphorylation sites, including residues 157 and 239 (UniProt P50552 modified residues). These annotations do not establish whether an unspecified antibody detects total or phosphorylated VASP; interpret phospho-specific staining only when the antibody's stated specificity supports it (general IHC practice). |
| Strength of the reference pattern | The HPA tissue profile is rated Enhanced while its staining-to-RNA consistency is described as medium (HPA tissue IHC). HPA also lists two antibodies with Enhanced IHC validation (HPA antibodies). Use these observations as reference patterns, while checking unexpected staining with appropriate controls (general IHC practice). |
| IF/ICC Q: Where should VASP appear? | A: Mainly at the plasma membrane, cell junctions and focal adhesion sites in the HPA ICC-IF images (HPA subcellular). UniProt additionally places VASP at lamellipodia and filopodial tips (UniProt P50552 subcellular location). This localization answer does not specify an IF/ICC protocol. |
| Situation | Likely cause | Next action |
|---|---|---|
| The positive-reference section has no VASP signal (HPA tissue IHC). | The expected cells may be absent from the section, or a staining step may have failed (general IHC practice). | Confirm cell identity and run a known-positive section through the same IHC workflow; review retrieval, primary incubation and detection records if both are blank (general IHC practice). |
| Staining is weak only in glia, fibroblasts or adipocytes (HPA tissue IHC). | HPA reports low staining in these populations (HPA tissue IHC). | Compare with an HPA high-staining cell population before changing conditions; low staining here alone does not show that the assay failed (HPA tissue IHC; general IHC practice). |
| The section shows diffuse color without clear cellular boundaries. | Background from the staining or detection workflow is possible (general IHC practice). | Compare a section processed without primary antibody, then review blocking, washes and chromogen development before scoring VASP (general IHC practice). |
| Cardiomyocytes or skeletal myocytes stain strongly (HPA tissue IHC). | HPA reports VASP as not detected in these cells; cell misidentification, cross-reactivity or endogenous detection activity are possibilities (HPA tissue IHC; general IHC practice). | Verify the stained cell type and compare reagent and detection controls before calling the signal VASP (general IHC practice). |
| Signal is mainly nuclear or extracellular. | That pattern conflicts with the reported cytoplasmic tissue profile and annotated cellular locations (HPA tissue IHC; UniProt P50552 subcellular location). | Recheck morphology and controls, and score only staining that can be assigned to a credible cellular compartment (general IHC practice). |
| Results differ between two sections that contain different cell populations. | HPA reports both high- and low-staining populations and low tissue specificity at the RNA level (HPA tissue IHC). | Compare the same identified cell population across sections and record compartment and intensity separately; do not treat unlike cell populations as equivalent controls (general IHC practice). |
Experiment evidence
IHC & IF images showing VASP staining
M00303 · IHCIHC analysis of VASP using anti-VASP antibody (M00303). VASP was detected in a paraffin-embedded section of human liver cancer tissue. Heat
M00303 · IHCIHC analysis of VASP using anti-VASP antibody (M00303). VASP was detected in a paraffin-embedded section of human liver tissue. Heat mediate
A00303-1 · IHCIHC analysis of VASP using anti-VASP antibody (A00303-1). VASP was detected in a paraffin-embedded section of rat intestine tissue. Heat med
M00303-3 · IHCHuman kidney was stained with anti-VASP rabbit antibody
M00303-3 · IHCHuman placenta was stained with anti-VASP rabbit antibody
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000125753-VASP/tissue/cerebral+cortex#imid_1751700
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000125753-VASP/tissue/colon#imid_2254990
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000125753-VASP/tissue/liver#imid_1751627
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000125753-VASP/tissue/kidney#imid_2254992
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody CAB004612; green = VASP, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody CAB004612; green = VASP, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for VASP IHC & IF
🧪Run appendix first: glandular cells should stain (HPA: Appendix glandular cells, High). Use heart muscle cardiomyocytes as the negative tissue (HPA: Not detected); on the appendix slide, treat only demonstrably unstained nonglandular cells as internal negatives, since the HPA row does not establish their VASP status.
Positive control tissue: Appendix (Glandular cells, HPA High)
Negative control tissue: Heart muscle (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show VASP in A-431, U-251MG, U2OS, U2OS, siRNA 1, U2OS, scrambled, with annotated localisation: Plasma membrane (supported), Cell Junctions (supported), Focal adhesion sites (supported) (HPA subcellular).
Technical controls: Include a no-primary (secondary-only) control, an immunoglobulin control matched to the primary antibody’s host, isotype and clonality, and a VASP-knockout biological control if available (caption: rabbit anti-VASP M00303; standard IHC controls). Quench endogenous peroxidase and assess background before interpreting appendix DAB staining (caption: HRP/DAB detection; standard IHC practice).
⚠️Feasibility: No target-specific fixation window or fixation effect is reported, and the selected M00303 paraffin-section caption does not report a fixative. The reported IHC method uses heat retrieval in EDTA at pH 8.0, but the evidence does not establish whether retrieval is required or whether frozen sections or IF would be easier (caption: EDTA heat retrieval; HPA: ICC-IF images). In appendix, check for peroxidase-related background when scoring glandular staining (HPA: Appendix glandular cells, High; caption: HRP/DAB detection; standard IHC practice).
HPA tissue IHC evidence for VASP
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — Medium consistency between antibody staining and RNA expression data.). Rows are taken directly from the HPA tissue chart — click any row's HPA link to view the source.
Positive expression · recommended positive controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Appendix | Glandular cells | High | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | High | Protein (IHC) | HPA → |
| Breast | Glandular cells | High | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | High | Protein (IHC) | HPA → |
| Cervix | Glandular cells | High | Protein (IHC) | HPA → |
Section 3
Advanced VASP IHC Tips
Troubleshooting guidance for chromogenic VASP IHC in paraffin sections, with a separate note on IF (caption M00303).
How should I troubleshoot weak VASP staining after antigen retrieval?
Start with heat-mediated antigen retrieval in EDTA at pH 8.0 (datasheet M00303). The documented paraffin-section example then used 10% goat serum and antibody at 1:50 overnight at 4°C (caption M00303). If staining is weak, check that heating was uniform and that sections remained attached before changing antibody concentration (standard IHC practice). Compare a known positive compartment on the same staining run, such as glandular cells in appendix, with the study sections (HPA: High in appendix glandular cells). Record retrieval conditions for every run because a stronger DAB signal alone does not establish greater VASP abundance (standard IHC practice).
Could fixation explain variable VASP staining between paraffin blocks?
Target-specific sensitivity of VASP to fixation is unknown from the supplied evidence; the selected paraffin-section caption does not state a fixative (caption M00303). Record each block’s fixative and fixation duration before comparing staining intensity across blocks (standard IHC practice). Keep section thickness, EDTA retrieval at pH 8.0, antibody incubation, and DAB development consistent when assessing variation (datasheet M00303; standard IHC practice). If blocks differ substantially, stain comparable positive-control tissue alongside them and interpret differences cautiously (standard IHC practice). Do not assign a fixation effect to VASP solely from its phosphorylation sites or tissue-expression pattern (UniProt P50552 modified residues; HPA tissue IHC).
Where should VASP staining appear in a paraffin section?
Expect predominantly cytoplasmic staining across many cell types in tissue sections (HPA: cytoplasmic expression in most cells). Junctional or peripheral enrichment can also fit VASP biology: it associates with focal adhesions, tight junctions, lamellipodia, and filopodial tips (UniProt P50552 subcellular location). Its supported plasma-membrane, junctional, and focal-adhesion locations come from subcellular imaging and should guide inspection of cellular borders (HPA subcellular). Compare the pattern with tissue morphology and a control section before calling a sharply confined nuclear signal positive (standard IHC practice; UniProt P50552 subcellular location). Platelets may contribute signal in a specimen because VASP is highly expressed in them (UniProt P50552 tissue specificity).
How can epitope choice affect interpretation of VASP IHC?
The supplied record lists 0 isoforms, so an apparent staining difference should not be attributed to an annotated VASP isoform here (UniProt P50552 isoforms). VASP has no transmembrane segment, and its documented locations include cytoplasm and membrane-associated structures (UniProt P50552 topology; UniProt P50552 subcellular location). It also has multiple modified residues, including phosphorylation at positions 157 and 239 (UniProt P50552 modified residues). Check whether the chosen antibody recognises total VASP or a specified modified epitope before comparing samples (standard IHC practice). The supplied caption gives staining conditions but no epitope map, so it cannot establish phosphorylation specificity (caption M00303).
How should I adapt these VASP observations for multiplex IF?
Use the separate IF workflow and verify its antibody conditions independently; the 1:50 dilution comes from paraffin-section chromogenic IHC (caption M00303). Multiplex VASP with a marker for the expected cell type, such as a glandular-cell marker when assessing appendix glands, and check whether the signals occupy the same cells (HPA: High in appendix glandular cells; standard IF practice). Choose fluorophores and acquisition channels after inspecting unstained tissue for autofluorescence (standard IF practice). Because VASP has no transmembrane segment and can occupy cytoplasmic and junctional sites, optimise permeabilisation to expose the relevant intracellular epitope (UniProt P50552 topology and subcellular location; standard IF practice). Include single-channel controls to assess bleed-through (standard IF practice).
What should I check when VASP DAB staining looks diffuse or excessive?
First inspect a no-primary control for secondary-reagent binding and endogenous peroxidase signal (standard IHC practice). Use a peroxidase block and consistent DAB development when troubleshooting the chromogenic workflow; these are general IHC steps, not evidence of VASP-specific behaviour (standard IHC practice). The selected example used 10% goat serum, 1:50 primary antibody overnight at 4°C, and a peroxidase-conjugated secondary for 30 minutes at 37°C (caption M00303). If background remains high, titrate antibody and compare with a matched control section (standard IHC practice). Preserve cellular context: widespread cytoplasmic VASP staining can be biological (HPA: cytoplasmic expression in most cells).
How should I score VASP staining across tissue sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring, because VASP may appear cytoplasmic and at cell borders (HPA tissue IHC; UniProt P50552 subcellular location). For chromogenic sections, report the percentage of positive cells and intensity, or calculate an H-score from intensity categories 0–3 for a range of 0–300 (standard IHC practice). If counting positive cells by area, report density per mm² of viable, annotated tissue (standard IHC practice). Normalise comparisons to the same cell population and scored area, with matched staining and imaging conditions (standard IHC practice). Report junctional enrichment separately from overall cytoplasmic intensity when that distinction matters (UniProt P50552 subcellular location; standard IHC practice).
How can I distinguish genuine VASP staining from artefact?
Look for cellular staining that fits VASP’s cytoplasmic, junctional, or focal-adhesion distribution (HPA tissue IHC; UniProt P50552 subcellular location). Check the stained cell type against morphology: appendix glandular cells and colon endothelial cells are reported as high, whereas heart cardiomyocytes are reported as not detected (HPA tissue IHC). Treat staining limited to section edges or necrotic areas cautiously, and compare it with intact tissue in the same section (standard IHC practice). A no-primary control helps reveal endogenous enzyme activity or detection-reagent background in DAB assays (standard IHC practice). Interpret intensity differences alongside controls because the HPA tissue profile has medium consistency with RNA expression (HPA: Enhanced reliability description).
Boster reagents
Best VASP / Vasodilator-stimulated phosphoprotein IHC Antibodies
The catalog includes VASP antibodies with IHC images from human liver cancer, rat intestine and human kidney (product image captions); one antibody also lists IF for human samples (M00303-3 catalog).
Real IHC data
Anti-VASP Rabbit Monoclonal Antibody
Cat # M00303
Real IHC data
Anti-VASP Antibody ®
Cat # A00303-1
Real IHC data
Anti-VASP Rabbit Monoclonal Antibody
Cat # M00303-3
M00303 is listed for human IHC, with a paraffin-section image from human liver cancer tissue (M00303 catalog and image caption); A00303-1 is listed for human and rat IHC, with a paraffin-section image from rat intestine (A00303-1 catalog and image caption). M00303-3 is listed for human IHC and IF, with an IHC image caption from human kidney and no IF image in the payload (M00303-3 catalog and image captions).
Which to pick: For human tissue IHC in paraffin sections, choose the rabbit monoclonal M00303: its own caption documents that sample preparation and 1:50 primary antibody dilution (M00303 catalog and image caption). For an IF experiment, choose M00303-3 because IF is listed for human samples at 1:200–1:1000; its image caption gives no processing details, and the payload provides no IF image (M00303-3 catalog and image captions). For human or rat IHC, choose A00303-1 based on its listed reactivity and rat-intestine paraffin-section image; the captions for M00303 and A00303-1 do not report a fixative (A00303-1 catalog and image caption; M00303 image caption).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P50552 (VASP_HUMAN, Vasodilator-stimulated phosphoprotein).
- Human Protein Atlas. VASP tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. VASP subcellular location (ICC-IF): Mainly localized to the plasma membrane, cell junctions and focal adhesion sites..
- Human Protein Atlas. VASP antibody validation summary (2 antibodies).
- A functional antiplatelet nanomotor for tri-modal ablation of acute arterial thrombus. Cell reports. Medicine 2026 — PMC13522756.
- Tumor suppressor berberine binds VASP to inhibit cell migration in basal-like breast cancer. Oncotarget 2016 — PMC5216765.
- Vasodilator-Stimulated Phosphoprotein Biomarkers Are Associated with Invasion and Metastasis in Colorectal Cancer. Biomarkers in cancer 2018 — PMC6419247.
- Dai Huang Fu Zi Tang could ameliorate intestinal injury in a rat model of hemorrhagic shock by regulating intestinal blood flow and intestinal expression of p-VASP and ZO-1. BMC complementary and alternative medicine 2014 — PMC3974027.
- PubMed PMID:7828592 — UniProt-cited evidence.
- PubMed PMID:10087267 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for VASP (Vasodilator-stimulated phosphoprotein). Expected localisation Cytoplasm in most cells (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet M00303). Validated IHC antibody: M00303 at 1:50 (the product's own IHC validation image is shown). Controls (HPA): positive Appendix (Glandular 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 M00303); 2. Fixation: Image fixative and duration unreported (datasheet M00303); 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 M00303); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet M00303); 8. Primary antibody: Rabbit monoclonal (clone BHO-22) anti-VASP, 1:50 (datasheet M00303); 9. Primary incubation: Overnight at 4 °C (datasheet M00303). Watch for: Fixation: Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image M00303); Caveat: Widespread staining limits cell-type specificity (HPA tissue IHC); Isoform / epitope: No annotated isoforms; chain 2–380 has no extracellular domain (UniProt); Regulation: Thrombin/PMA recruit VASP to focal adhesions (UniProt). Evidence: UniProt P50552, HPA tissue IHC · Enhanced, HPA ICC-IF · Mainly localized to the plasma membrane, cell junctions and focal…, Datasheet M00303, PMC5216765, PMC6419247. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/VASP/immunohistochemistry-ihc-guide.