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VPS4A / Vacuolar protein sorting-associated protein 4A · IHC design guide
Design Immunohistochemistry for VPS4A
Plan VPS4A paraffin IHC around the reported widespread cytoplasmic pattern (HPA tissue IHC). This guide covers fixation, controls and interpretation; staining has low consistency with RNA expression (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
VPS4A 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 staining in tissue (HPA tissue IHC) | |
| Staining pattern | Widespread cytoplasmic staining across cell types (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A03216-1) | |
| Positive control | Adrenal gland+4 more · see all | |
| Negative control | None in HPA (detected in all 45 tissues); use no-primary + isotype controls |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent across sections. (standard IHC practice; not target-specific) | |
| Caveat | Staining has low consistency with RNA expression (HPA tissue IHC) | |
| Regulation | Ubiquitously expressed (UniProt) | |
| Isoform / epitope | 0 annotated isoforms; one 1–437 chain (UniProt) |
Section 1
Recommended VPS4A IHC & IF Protocols
Compare the catalog antibody’s EDTA pH 8.0 IHC-P protocol (datasheet A03216-1) with published VPS4A IHC methods for human colon arrays (PMC7005644) and dewaxed sections (PMC12190599).
| Sample | Paraffin-embedded human lung cancer tissue; fixative not specified (datasheet A03216-1) |
| Fixation | Image fixative and duration unreported (datasheet A03216-1); 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 A03216-1); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A03216-1) |
| Primary antibody | Rabbit anti-VPS4A, 0.5-1μg/ml (datasheet A03216-1) |
| Primary incubation | Overnight at 4 °C (datasheet A03216-1) |
| Detection | Streptavidin-biotin complex (SABC), DAB chromogen (datasheet A03216-1) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | VPS4A-positive staining in glandular cells of adrenal gland (HPA tissue IHC: Medium). HPA tissue profile: Ubiquitous cytoplasmic expression. No signal in the no-primary control. |
💡Decision noteStart with EDTA pH 8.0 heat retrieval for the catalog antibody (datasheet A03216-1). Citrate pH 6.0 was used with a different VPS4A antibody (PMC12190599).
Section 2
What Is the Expected VPS4A Staining Pattern?
VPS4A should appear mainly cytoplasmic across many cell types in paraffin IHC; dividing cells may also show midbody-associated signal (HPA tissue IHC: ubiquitous cytoplasmic expression; UniProt Q9UN37: midbody). It associates with late endosome membranes but has no transmembrane segment (UniProt Q9UN37 topology). Treat the tissue pattern as a guide, not definitive proof: HPA rates its IHC evidence Approved while reporting low consistency between staining and RNA expression (HPA tissue IHC).
| Cytoplasmic staining in glandular or neuronal cells, with variable intensity across the section. | This fits the broad pattern (HPA tissue IHC: ubiquitous cytoplasmic expression). HPA reports Medium staining in glandular cells of adrenal gland, appendix, breast and cervix, and in neuronal cells of caudate and cerebral cortex (HPA tissue IHC). Judge compartment and cell identity together; the HPA reliability caveat still applies (HPA tissue IHC: Approved, low RNA–staining consistency). |
| A sharply defined midbody signal in an identifiable dividing cell. | This is biologically plausible: VPS4A localizes to the midbody during cytokinesis, including rings on either side of the Flemming body (UniProt Q9UN37). Routine chromogenic sections may not resolve those rings (general IHC practice). Do not require visible midbodies in every field; their appearance depends on capturing cells at the relevant division stage (UniProt Q9UN37; general IHC practice). |
| Strong, predominantly nuclear or surface-restricted staining, with little cytoplasmic signal. | Treat this as a compartment mismatch requiring investigation: the reported locations are cytoplasm, late endosome membrane and midbody (UniProt Q9UN37; HPA tissue IHC: cytoplasmic). Check morphology, counterstain and detection controls before assigning VPS4A specificity (general IHC practice). A focal endosome-associated pattern is plausible; uniform cell-surface staining is not an expected pattern from these records (UniProt Q9UN37). |
| Intense staining limited to a cell population expected to stain weakly. | Compare like-for-like cells before calling cross-reactivity: HPA lists Low staining in cholangiocytes, trophoblastic cells, cardiomyocytes, smooth muscle cells, myocytes and adipocytes, but lists no negative tissue (HPA tissue IHC). Strong selective staining in one of these populations warrants checks for nonspecific antibody binding or endogenous detection activity (general IHC practice), not an automatic biological exclusion. |
| Haze throughout tissue, or no signal in a section containing an HPA Medium cell population. | Diffuse haze obscures the expected cytoplasmic pattern (HPA tissue IHC; general IHC practice). Absent signal in, for example, appendix glandular cells calls for a control and workflow check (HPA tissue IHC: Medium in appendix glandular cells; general IHC practice). A single negative section cannot establish true absence, particularly given HPA's low RNA–staining consistency (HPA tissue IHC). |
💡Expected VPS4A appearanceCall a result consistent with VPS4A when predominantly cytoplasmic staining is visible at roughly Medium intensity in an HPA-listed glandular or neuronal cell population, allowing occasional midbody signal in dividing cells; dominant nuclear staining or uniform tissue-wide haze should trigger control review (HPA tissue IHC: Medium populations and cytoplasmic profile; UniProt Q9UN37: midbody; general IHC practice).
| Subcellular context | VPS4A is membrane-associated at late endosomes despite lacking a transmembrane segment (UniProt Q9UN37 topology). Expect cytoplasmic staining in tissue IHC (HPA tissue IHC); do not score it as a continuous plasma membrane rim (UniProt Q9UN37; general IHC practice). |
| Tissue selection and evidence strength | HPA reports Medium staining in several glandular and neuronal populations and Low staining in the listed muscle, adipose, placental and cholangiocyte populations (HPA tissue IHC). It lists no negative tissue and notes low consistency with RNA expression (HPA tissue IHC), so use a Medium population as a practical positive reference without treating a Low population as a knockout control. |
| Antibody validation | HPA lists IHC as Approved for CAB018751 and CAB034411, while HPA060092 has ICC Approved and no listed IHC status (HPA antibodies). Approved should not be read as Enhanced; interpret staining with the reported tissue reliability caveat (HPA antibodies; HPA tissue IHC). |
| Protein form | UniProt lists one 437-aa chain, no signal peptide or propeptide, no listed isoforms and no transmembrane segment (UniProt Q9UN37). These annotations give no basis to expect a secreted, shed or tissue-specific processed staining compartment; they do not establish how antigen retrieval affects this antibody. |
| IF/ICC Q: what pattern should I expect? | A: HPA supports midbody localization in ICC-IF images, with a caution that the subcellular summary uses antibodies targeting proteins from multiple genes (HPA subcellular). UniProt also places VPS4A at the midbody (UniProt Q9UN37). Interpret IF/ICC against its own image and antibody evidence; the IHC tissue pattern does not establish an IF/ICC protocol (HPA subcellular; HPA antibodies). |
| Situation | Likely cause | Next action |
|---|---|---|
| No stain in an HPA Medium reference population. | Target-specific fixation effects are not established by the supplied assay evidence. Verify with a matched IHC source before attributing a result to fixation. | Confirm tissue identity and run a working positive control; review retrieval, primary incubation and detection against the antibody's own IHC instructions (general IHC practice). |
| Diffuse brown deposit or broad tissue haze. | Nonspecific primary binding, residual endogenous enzyme activity or detection background can obscure cytoplasmic signal (general chromogenic IHC practice). | Inspect a primary-omission control; check blocking and washes, and apply the detection system's endogenous-activity controls as appropriate (general IHC practice). |
| Strong nuclear stain dominates. | This differs from the reported cytoplasmic and midbody locations (HPA tissue IHC; UniProt Q9UN37). | Verify cell boundaries with the counterstain and compare an IHC-validated antibody plus appropriate negative controls before scoring the nucleus as VPS4A (HPA antibodies; general IHC practice). |
| Low-listed cells stain more strongly than nearby Medium-listed cells. | The result conflicts with the reported tissue comparison, although HPA's staining–RNA consistency is low (HPA tissue IHC). | Confirm cell identification and compare matched sections; review primary-omission and endogenous-detection controls before claiming a new tissue pattern (general IHC practice). |
| No visible midbody signal despite cytoplasmic staining. | Midbody localization occurs during division, and a section may contain no resolvable midbody at that stage (UniProt Q9UN37; general IHC practice). | Score the cytoplasmic tissue pattern first; assess midbody signal only in clearly dividing cells with suitable morphology (HPA tissue IHC; UniProt Q9UN37; general IHC practice). |
| ICC-Approved antibody gives uncertain paraffin IHC staining. | HPA lists ICC Approved but no IHC status for HPA060092; its ICC result does not establish paraffin IHC performance (HPA antibodies). | Use an antibody with listed IHC approval, such as CAB018751 or CAB034411, and assess its controls and tissue pattern (HPA antibodies; HPA tissue IHC; general IHC practice). |
Experiment evidence
IHC & IF images showing VPS4A staining
A03216-1 · IHCIHC analysis of VPS4A using anti-VPS4A antibody (A03216-1). VPS4A was detected in paraffin-embedded section of human lung cancer tissue. Hea
A03216-1 · IHCIHC analysis of VPS4A using anti-VPS4A antibody (A03216-1). VPS4A was detected in paraffin-embedded section of human mammary cancer tissue.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000132612-VPS4A/tissue/cerebral+cortex#imid_15665647
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000132612-VPS4A/tissue/colon#imid_15664865
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000132612-VPS4A/tissue/liver#imid_15664866
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000132612-VPS4A/tissue/kidney#imid_15664859
HPA · ICC-IF SiHaHuman Protein Atlas immunofluorescence, SiHa, antibody HPA060092; green = VPS4A, blue = nucleus, red = microtubules.
HPA · ICC-IF SiHaHuman Protein Atlas immunofluorescence, SiHa, antibody HPA060092; green = VPS4A, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for VPS4A IHC & IF
🧪Run bronchus first and score respiratory epithelial cells, which show Medium staining (HPA: bronchus, respiratory epithelial cells, Medium). HPA detects VPS4A in all 45 scored tissues, so there is no supported negative tissue or prespecified negative cell population within the positive slide; use no-primary and isotype controls to assess background, and interpret any unstained cells by their morphology rather than assuming they are VPS4A-negative (HPA: no negative rows; detected in all 45 scored tissues).
Positive control tissue: Adrenal gland (Glandular cells, HPA Medium)
Negative control tissue: None in HPA: VPS4A is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead.
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show VPS4A in SiHa, U2OS, hTERT-RPE1 (serum starved), KOLF2.1J, with annotated localisation: Midbody (supported) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only section and a concentration-matched rabbit isotype control, then confirm specificity with VPS4A knockout material or an antigen peptide block if available (caption: rabbit primary antibody; standard IHC controls). For the caption’s biotin-based DAB detection, quench endogenous peroxidase and check for endogenous biotin background; assess bronchial tissue autofluorescence if using IF (caption: biotinylated secondary, SABC and DAB; standard IHC/IF controls).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected A03216-1 paraffin-section caption does not state the fixative (selected-SKU tissue-IHC caption). That caption used heat retrieval in EDTA, pH 8.0, but it does not establish that retrieval is required; the supplied evidence does not show that frozen sections or IF are easier (selected-SKU tissue-IHC caption). In bronchus, mucus or tissue autofluorescence can complicate interpretation, so compare signal with the matched control section (standard IHC/IF practice).
HPA tissue IHC evidence for VPS4A
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 |
|---|---|---|---|---|
| Adrenal gland | Glandular cells | Medium | Protein (IHC) | HPA → |
| Appendix | Glandular cells | Medium | Protein (IHC) | HPA → |
| Breast | Glandular cells | Medium | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | Medium | Protein (IHC) | HPA → |
| Caudate | Neuronal cells | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| None in HPA: VPS4A is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead. | ||||
Section 3
Advanced VPS4A IHC Tips
Troubleshoot VPS4A staining in paraffin sections by checking retrieval, cellular location, controls, and scoring before interpreting signal intensity.
Which retrieval conditions should I use when VPS4A staining is weak in paraffin sections?
Start with heat-mediated antigen retrieval in EDTA at pH 8.0 for paraffin sections stained with A03216-1 (datasheet A03216-1). The selected tissue image used this retrieval before incubation with 1 µg/ml primary antibody overnight at 4°C (datasheet A03216-1). If signal is weak, compare adjacent sections while changing heating duration incrementally, and keep antibody concentration and detection conditions constant (standard IHC practice). Include a known staining section and a no-primary control in each run so retrieval changes can be distinguished from detection background (standard IHC practice). Record whether improved signal remains predominantly cytoplasmic, as expected in tissue IHC (HPA: ubiquitous cytoplasmic expression).
Could fixation explain inconsistent VPS4A staining between paraffin blocks?
The selected paraffin-section caption does not report a fixative, so VPS4A-specific fixation sensitivity is unknown for this antibody (datasheet A03216-1). Record each block’s fixative and fixation duration before comparing staining, because processing differences can change epitope accessibility in IHC (standard IHC practice). Run sections from differently processed blocks together using EDTA retrieval at pH 8.0 and the same detection reagents (datasheet A03216-1; standard IHC practice). If one block stains weakly, inspect tissue preservation and compare an independent control antigen before attributing the difference to VPS4A abundance (standard IHC practice). Do not infer fixation tolerance from cytoplasmic expression or protein topology (HPA: tissue IHC; UniProt Q9UN37 topology).
Where should VPS4A signal appear in chromogenic tissue sections?
Expect predominantly cytoplasmic tissue staining, while allowing discrete signal associated with endosomal membranes or dividing-cell midbodies (HPA: tissue IHC; UniProt Q9UN37 subcellular location). VPS4A associates with late endosome membranes despite having no transmembrane segment, so membrane-adjacent puncta need cellular context rather than a surface-membrane score (UniProt Q9UN37 topology and location). Its reported midbody localisation includes rings beside the Flemming body, a pattern that may be difficult to resolve with chromogenic tissue IHC (UniProt Q9UN37 subcellular location). Compare candidate puncta with cell morphology and adjacent sections before assigning them to a specific structure (standard IHC practice). Treat diffuse nuclear-only staining as discordant with the supplied localisation evidence and investigate controls (HPA: tissue IHC; UniProt Q9UN37 location).
How should I assess epitope access and possible VPS4A cross-reactivity?
The supplied record lists 0 isoforms and one 437-residue chain, so an isoform-specific staining explanation lacks support here (UniProt Q9UN37 isoforms and processing). The record places the MIT domain at residues 2–80 and reports modifications including phosphoserines 95 and 97, but the antibody epitope is unspecified (UniProt Q9UN37 domains and modified residues; datasheet A03216-1). Do not claim that retrieval exposes a particular domain or that phosphorylation changes binding without epitope-mapping evidence (standard IHC interpretation). Check antibody documentation for immunogen sequence and assess similarity to VPS4B before treating ambiguous staining as VPS4A-specific (UniProt Q9UN37: VPS4B interaction; standard IHC practice). An independent antibody against a documented, distinct epitope can strengthen localisation findings (standard IHC practice).
How can IF help assess the VPS4A pattern seen by chromogenic IHC?
Use IF as a separate localisation check, pairing VPS4A with a marker for the expected cell type when mixed populations complicate IHC interpretation (standard IF practice). Choose fluorophores after examining tissue autofluorescence, and use single-label controls to assess channel bleed-through before interpreting apparent overlap (standard IF practice). Because VPS4A is membrane-associated without a transmembrane segment, choose permeabilisation conditions that permit antibody access to its intracellular epitope, once that epitope is known (UniProt Q9UN37 topology and location; standard IF practice). Midbody enrichment is supported in subcellular images, but that source cautions that its antibodies target proteins from multiple genes (HPA: subcellular). Concordant IF and IHC patterns support interpretation without establishing catalog-antibody IF validation (standard assay interpretation).
What should I check when VPS4A IHC has widespread brown background?
First inspect a no-primary section to separate detection background from signal associated with the primary antibody (standard IHC practice). The selected image used 10% goat serum blocking, biotinylated goat anti-rabbit secondary antibody, a streptavidin-biotin complex, and DAB (datasheet A03216-1). Check endogenous peroxidase blocking and, for this biotin-based detection system, assess endogenous biotin if background persists (standard chromogenic IHC practice). Compare background at tissue edges and damaged regions with well-preserved interior tissue before adjusting antibody concentration (standard IHC practice). Because VPS4A has broadly reported cytoplasmic tissue expression, widespread staining alone cannot establish specificity; require appropriate cellular detail and controls (HPA: tissue IHC; standard IHC practice).
How should I quantify VPS4A staining across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and cytoplasmic compartment before scoring, consistent with the reported tissue IHC pattern (HPA: ubiquitous cytoplasmic expression; standard IHC practice). For intensity differences, report an H-score with 0–3 intensity categories and the percentage of cells in each category; retain the raw percentages (standard IHC practice). Alternatively, report percent positive cells or positive-cell density per mm² when intensity grading is unreliable (standard IHC practice). Normalize counts to viable, evaluable tissue area or the number of cells in the prespecified population, and apply one threshold across slides (standard IHC practice). Analyze runs with matched staining and imaging conditions because the HPA tissue profile has low agreement with RNA expression (HPA: Approved, low consistency).
How can I distinguish a true VPS4A-positive cell from staining artefact?
A plausible positive shows cytoplasmic staining with preserved cell boundaries; a discrete midbody-associated signal may appear in dividing cells (HPA: tissue IHC; UniProt Q9UN37 subcellular location). Check whether the stained cell population matches the morphology being scored, since the reported tissue profile is broadly cytoplasmic rather than restricted to one tissue (HPA: tissue IHC). Discount staining confined to section edges, necrotic areas, or sites also positive in no-primary controls, and check endogenous peroxidase activity (standard IHC practice). Predominantly nuclear-only signal should prompt review of controls and localisation before a VPS4A assignment (UniProt Q9UN37 location; standard IHC practice). Interpret modest intensity differences cautiously because HPA reports low consistency between antibody staining and RNA expression (HPA: Approved reliability description).
Boster reagents
Best VPS4A / Vacuolar protein sorting-associated protein 4A IHC Antibodies
A03216-1 has real IHC images from human paraffin sections of lung and mammary cancer tissue (catalog IHC captions); no IF image is supplied (catalog IF images).
Real IHC data
Anti-VPS4A Antibody ®
Cat # A03216-1
A03216-1 is listed for IHC in human, mouse and rat samples (catalog applications and reactivity). Its IHC images show human lung and mammary cancer paraffin sections (catalog IHC captions).
Which to pick: Choose A03216-1 for tissue IHC: its rabbit antibody has an IHC application listing and images from human paraffin sections (catalog host, applications and IHC captions). Its listed reactivity includes mouse and rat, but the supplied IHC images show human tissue only (catalog reactivity and IHC captions). No SKU has supplied IF/ICC validation, so this catalog does not support an IF/ICC pick; the fixative used for the A03216-1 paraffin sections is unreported (catalog applications, IF images and IHC captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q9UN37 (VPS4A_HUMAN, Vacuolar protein sorting-associated protein 4A).
- Human Protein Atlas. VPS4A tissue IHC expression (reliability: Approved).
- Human Protein Atlas. VPS4A subcellular location (ICC-IF): Localized to the midbody. Caution: Based on antibodies targeting proteins from multiple genes..
- Human Protein Atlas. VPS4A antibody validation summary (3 antibodies).
- Vps4A functions as a tumor suppressor by regulating the secretion and uptake of exosomal microRNAs in human hepatoma cells. Hepatology (Baltimore, Md.) 2015 — PMC4511093.
- Synthetic lethality between VPS4A and VPS4B triggers an inflammatory response in colorectal cancer. EMBO molecular medicine 2020 — PMC7005644.
- A Novel Modulator of Resistance for Oxaliplatin-Based Therapy for Colorectal Cancer: The ESCRT Family Member VPS4A. Cells 2025 — PMC12190599.
- PubMed PMID:11563910 — UniProt-cited evidence.
- PubMed PMID:12594041 — UniProt-cited evidence.
- PubMed PMID:10931946 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for VPS4A (Vacuolar protein sorting-associated protein 4A). Expected localisation Cytoplasmic staining in tissue (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A03216-1). Validated IHC antibody: A03216-1 at 0.5-1μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Adrenal gland (Glandular cells); negative None in HPA (detected in all 45 tissues); use no-primary + isotype controls. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human lung cancer tissue; fixative not specified (datasheet A03216-1); 2. Fixation: Image fixative and duration unreported (datasheet A03216-1); 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 A03216-1); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A03216-1); 8. Primary antibody: Rabbit anti-VPS4A, 0.5-1μg/ml (datasheet A03216-1); 9. Primary incubation: Overnight at 4 °C (datasheet A03216-1). Watch for: Fixation: Keep fixation consistent across sections. (standard IHC practice; not target-specific); Caveat: Staining has low consistency with RNA expression (HPA tissue IHC); Isoform / epitope: 0 annotated isoforms; one 1–437 chain (UniProt); Regulation: Ubiquitously expressed (UniProt). Evidence: UniProt Q9UN37, HPA tissue IHC · Approved, HPA ICC-IF · midbody. Caution: Based on antibodies targeting proteins from…, Datasheet A03216-1, PMC7005644, PMC12190599. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/VPS4A/immunohistochemistry-ihc-guide.