VPS4B / Vacuolar protein sorting-associated protein 4B · IHC design guide

Design Immunohistochemistry for VPS4B

Plan chromogenic VPS4B IHC on paraffin sections with the IHC-validated antibody (datasheet A03403-1). Compare cytoplasmic staining in esophageal squamous cells with alveolar cells scored as not detected (HPA tissue IHC).

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

Immunohistochemistry protocol sheet for VPS4B (IHC for VPS4B): expected localisation Cytoplasmic tissue staining (HPA tissue IHC); late-endosome membrane association (UniProt), antibody A03403-1, validated IHC image, and IHC protocol steps — Printable **VPS4B** IHC protocol sheet — expected localisation Cytoplasmic tissue staining (HPA tissue IHC); late-endosome membrane association (UniProt), antibody A03403-1, controls and protocol steps. Open the full VPS4B IHC guide →

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VPS4B 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 tissue staining (HPA tissue IHC); late-endosome membrane association (UniProt)
	Staining pattern	Cytoplasmic staining in esophageal squamous cells (HPA tissue IHC)
	Antigen retrieval	EDTA pH 8.0 HIER, heat-mediated (datasheet A03403-1)
	Positive control ⓘ	Bone marrow+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 consistent across paraffin sections. (standard IHC practice; not target-specific)
	Caveat	Staining has medium agreement with RNA expression (HPA tissue IHC)
	Regulation	No expression regulator specified (UniProt)
	Isoform / epitope	No listed isoforms; one uncleaved chain, no transmembrane segment (UniProt)

Section 1

Recommended VPS4B IHC & IF Protocols

The catalog antibody’s IHC-P protocol is accompanied by one published VPS4B IHC protocol using colorectal tissue and xenografts (PMC7005644 methods).

Recommended immunohistochemistry (IHC-P) protocol parameters
Sample	Paraffin-embedded human lung cancer tissue; fixative not specified (datasheet A03403-1)
Fixation	Image fixative and duration unreported (datasheet A03403-1); 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 A03403-1); 20 min, 95–100 °C (standard)
Peroxidase block	3% H2O2, 10 min, room temperature (standard)
Blocking	10% goat serum (datasheet A03403-1)
Primary antibody	Rabbit anti-VPS4B, 0.5-1μg/ml (datasheet A03403-1)
Primary incubation	Overnight at 4 °C (datasheet A03403-1)
Detection	Streptavidin-biotin complex (SABC), DAB chromogen (datasheet A03403-1)
Counterstain	Hematoxylin, blue, dehydrate and mount (standard)
Expected result	VPS4B-positive staining in hematopoietic cells of bone marrow (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression in several different tissues types, including esophagus. No signal in the no-primary control.

💡Decision noteStart with heat-mediated EDTA retrieval at pH 8.0 (A03403-1 datasheet); the published protocol does not state retrieval conditions (PMC7005644 methods).

Section 2

What Is the Expected VPS4B Staining Pattern?

VPS4B is membrane-associated in the late endosomal compartment but has no transmembrane segment (UniProt O75351 topology). Expect cytoplasmic staining in several tissue types, especially esophageal squamous epithelial cells (HPA tissue IHC: High). The tissue IHC profile is Approved, with medium consistency between staining and RNA expression; assess the observed cell population rather than assuming every cell is positive (HPA tissue IHC: reliability).

What am I looking at on my slide?
Cytoplasmic staining in esophageal squamous epithelial cells.	This fits the reported distribution and a High signal in those cells (HPA tissue IHC: esophagus). Chromogenic IHC need not resolve individual vesicles to agree with the mainly vesicular ICC-IF localisation (HPA subcellular: Vesicles).
Dominant nuclear staining or a continuous plasma-membrane rim.	These compartments do not match the reported late endosome association or mainly vesicular localisation (UniProt O75351 subcellular; HPA subcellular). Suspect an artefact or off-target signal; compare with a known-positive section and a no-primary control (general IHC practice).
Strong signal in a cell population reported as not detected.	For example, HPA reports no detection in adipocytes, not in every cell of adipose tissue (HPA tissue IHC: adipose tissue). Confirm cell identity, then check cross-reactivity and, with enzyme-based detection, endogenous activity using appropriate controls (general IHC practice).
Diffuse colour across tissue and blank areas.	Poorly confined colour is less persuasive than cell-associated cytoplasmic staining (HPA tissue IHC: profile). Check the no-primary control, blocking, washes and detection background before assigning it to VPS4B (general IHC practice).
No signal in esophageal squamous epithelial cells.	That conflicts with the reported High staining in this cell population (HPA tissue IHC: esophagus). Verify that the positive-control section developed, then review retrieval, antibody concentration and detection using the chosen IHC workflow (general IHC practice).

💡Expected VPS4B appearanceCall a section positive when identifiable squamous epithelial cells show cytoplasmic staining, potentially High in esophagus (HPA tissue IHC: esophagus); dominant nuclear colour or colour in blank areas warrants a control check (UniProt O75351 subcellular; general IHC practice).

How each factor affects the staining
Compartment and topology	VPS4B associates with the late endosomal membrane without spanning it (UniProt O75351 topology). HPA describes the tissue IHC pattern as cytoplasmic and the ICC-IF pattern as mainly vesicular (HPA tissue IHC; HPA subcellular).
Choice of comparison cells	Esophageal and cervical squamous epithelial cells, plus bone marrow hematopoietic cells, are reported High (HPA tissue IHC). Adipocytes and lung alveolar cells are reported Not detected; these labels apply to the named cells, not whole tissues (HPA tissue IHC).
Strength of tissue evidence	The HPA tissue IHC assessment is Approved but reports medium consistency with RNA expression (HPA tissue IHC: reliability). Treat an unexpected result as a reason to inspect controls and cell identity, rather than treating the atlas profile as an absolute threshold.
Antibody validation	HPA lists IHC as Approved for HPA057649 and CAB046445, while HPA040393 has ICC Approval only (HPA antibodies). Match the evidence to the application and antibody used; an ICC result alone does not validate paraffin-section staining (HPA antibodies).
IF/ICC: what appearance should I expect?	Mainly vesicular localisation is the approved ICC-IF observation (HPA subcellular). The listed image cell lines are HaCaT, RT-4, U2OS and KOLF2.1J (HPA subcellular); this is a localisation comparison, not an IHC protocol.
Isoforms and processing	The supplied UniProt record lists 0 isoforms, no signal peptide or propeptide, and a 1–444 chain (UniProt O75351 processing). It gives no basis for predicting a separate isoform-specific or shed-product staining pattern.

Why is my staining missing, weak or wrong?
Situation	Likely cause	Next action
A known-positive esophagus section is blank.	The observation differs from High staining in squamous epithelial cells (HPA tissue IHC: esophagus); the failed step cannot be identified from that result alone.	Check tissue preservation, retrieval, antibody incubation and detection with a positive control (general IHC practice). Do not infer VPS4B-specific fixation sensitivity from the HPA profile.
Only nuclei appear strongly coloured.	Nuclear dominance conflicts with late endosome association and the mainly vesicular ICC-IF localisation (UniProt O75351 subcellular; HPA subcellular).	Inspect the no-primary control and counterstain, then reassess antibody concentration and detection background (general IHC practice).
Colour spreads across tissue or blank slide areas.	The pattern does not match the reported cytoplasmic, cell-associated tissue signal (HPA tissue IHC: profile); diffuse deposition can arise during staining (general IHC practice).	Compare no-primary sections, review blocking and wash steps, and confirm that development was controlled (general IHC practice).
An apparent positive is in adipocytes.	Adipocytes are reported Not detected, although that statement does not describe every cell in adipose tissue (HPA tissue IHC: adipose tissue).	Confirm the stained cells are adipocytes, compare an esophageal positive control, and test for nonspecific or endogenous detection signal (general IHC practice).
Vesicles are not individually visible in chromogenic IHC.	The mainly vesicular call comes from ICC-IF; tissue IHC is described more broadly as cytoplasmic (HPA subcellular; HPA tissue IHC: profile).	Judge the compartment and cell type at the resolution of the chromogenic section; use the separate ICC-IF evidence when evaluating punctate localisation (HPA subcellular).
Different IHC antibodies give different patterns.	HPA records IHC Approval for HPA057649 and CAB046445, without an Enhanced IHC designation in the supplied record (HPA antibodies).	Compare the same cell populations and control sections, and document the antibody used before attributing discordance to biology (general IHC practice).

Source: UniProt O75351 Human Protein Atlas tissue Human Protein Atlas subcellular

Experiment evidence

IHC & IF images showing VPS4B staining

IHC analysis of VPS4B/MIG1 using anti-VPS4B/MIG1 antibody (A03403-1). VPS4B/MIG1 was detected in paraffin-embedded section of human lung cancer tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1μg/ml rabbit anti-VPS4B/MIG1 Antibody (A03403-1) overnight at 4°C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) (Catalog # SA1022) with DAB as the chromogen.

A03403-1 · IHCIHC analysis of VPS4B/MIG1 using anti-VPS4B/MIG1 antibody (A03403-1). VPS4B/MIG1 was detected in paraffin-embedded section of human lung can

HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000119541-VPS4B/tissue/cerebral+cortex#imid_12634997

HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000119541-VPS4B/tissue/colon#imid_12634931

HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000119541-VPS4B/tissue/liver#imid_12634932

HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000119541-VPS4B/tissue/kidney#imid_12634933

HPA · ICC-IF HaCaTHuman Protein Atlas immunofluorescence, HaCaT, antibody HPA040393; green = VPS4B, blue = nucleus, red = microtubules.

← swipe / scroll for more images →

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

Sample controls for VPS4B IHC & IF

🧪Run bone marrow first; hematopoietic cells should stain (HPA: High in bone marrow hematopoietic cells). Run adipose tissue as the negative, where adipocytes are not detected (HPA: Not detected in adipocytes); any cells treated as internal negatives on the marrow slide should show counterstain without specific chromogen, but the supplied HPA rows do not identify a negative marrow cell type.

Positive control tissue: Bone marrow (Hematopoietic cells, HPA High)

Negative control tissue: Adipose tissue (HPA Not detected)

ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show VPS4B in HaCaT, RT-4, U2OS, KOLF2.1J, with annotated localisation: Vesicles (approved) (HPA subcellular).

Technical controls: Include no-primary (secondary-only), a rabbit control immunoglobulin matched to the primary antibody’s isotype and clonality when known, and VPS4B knockout material or a validated peptide-block control (caption: rabbit primary antibody; standard IHC practice). Block endogenous peroxidase and assess endogenous biotin when using the caption’s biotin-based DAB detection in bone marrow (caption: biotinylated secondary and SABC–DAB; standard IHC practice).

⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected-SKU tissue-IHC caption does not state a fixative (caption: fixative not stated). The caption reports heat-mediated retrieval in EDTA at pH 8.0, but does not establish that retrieval is required; the supplied evidence does not establish whether frozen sections or IF are easier (caption: EDTA retrieval; HPA: ICC-IF images available). Bone marrow may show endogenous peroxidase signal, so interpret DAB staining against the no-primary control (standard IHC practice).

HPA tissue IHC evidence for VPS4B

Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — 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
Bone marrow	Hematopoietic cells	High	Protein (IHC)	HPA →
Cervix	Squamous epithelial cells	High	Protein (IHC)	HPA →
Esophagus	Squamous epithelial cells	High	Protein (IHC)	HPA →
Nasopharynx	Respiratory epithelial cells	High	Protein (IHC)	HPA →
Spleen	Cells in red pulp	High	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 →
Endometrium	Cells in endometrial stroma	Not detected	Protein (IHC)	HPA →
Epididymis	Glandular cells	Not detected	Protein (IHC)	HPA →
Gallbladder	Glandular cells	Not detected	Protein (IHC)	HPA →

Section 3

Advanced VPS4B IHC Tips

Troubleshoot VPS4B staining in paraffin sections by checking retrieval, cellular distribution, background and scoring against the supplied IHC and localization evidence.

Which retrieval conditions should I try first for weak VPS4B staining?

Start with heat-mediated retrieval in EDTA at pH 8.0 for the catalog antibody in paraffin sections (datasheet A03403-1). The documented tissue image used this retrieval before an overnight primary incubation at 4°C, so keep those conditions together while establishing a reference stain (caption A03403-1). If staining remains weak, compare a more vigorous heat exposure on adjacent sections while keeping the EDTA buffer and detection conditions constant (standard IHC practice). Check whether signal improves in cytoplasm or vesicle-like structures, since diffuse nuclear color would conflict with the reported localization (HPA: cytoplasmic tissue expression; HPA subcellular: vesicles approved).

Could fixation explain weak VPS4B staining in my paraffin sections?

Target-specific fixation sensitivity is unknown because the selected paraffin-section caption does not state a fixative (caption A03403-1: fixative not stated). Record each specimen’s fixative and time in fixative, then compare sections processed alike with the documented EDTA retrieval at pH 8.0 (datasheet A03403-1; standard IHC practice). If a batch is weak, inspect morphology and compare staining across specimens with known processing histories before changing antibody concentration (standard IHC practice). Do not use the reported tissue pattern to diagnose fixation damage; that pattern describes staining distribution, not VPS4B fixation sensitivity (HPA: cytoplasmic expression in several tissue types).

Where should convincing VPS4B signal appear in tissue IHC?

Expect predominantly cytoplasmic staining in tissue sections, with a vesicle-associated pattern possible where resolution permits (HPA: cytoplasmic tissue expression; HPA subcellular: vesicles approved). VPS4B associates with the late endosome membrane and has no transmembrane segment, so a sharp, continuous plasma-membrane outline is difficult to reconcile with its annotated location (UniProt O75351: subcellular location and topology). Compare the suspect compartment with hematoxylin-defined nuclei and neighboring cells at the same exposure and DAB development (standard IHC practice). If only nuclei or tissue edges stain, repeat with a no-primary control and assess whether the pattern persists (standard IHC practice).

How should I investigate an unexpected VPS4B staining pattern?

The supplied record lists 0 isoforms and a single 1–444 chain, so an unexpected compartment cannot be assigned to an annotated splice isoform here (UniProt O75351: isoforms and processing). The MIT domain spans residues 4–82, and four modified residues are listed, including phosphoserines at 93, 102, 108 and 410 (UniProt O75351: domains and modified residues). The catalog epitope is unspecified in the supplied evidence, so avoid claiming that retrieval exposes a particular domain or phosphorylation state (caption A03403-1: epitope not stated). Compare independent sections and a no-primary control, then seek epitope information before making a molecular interpretation (standard IHC practice).

How can IF help check an ambiguous VPS4B IHC pattern?

Use IF as a complementary localization check, starting with a marker for the expected cell population in the tissue under study (standard IF practice). Choose fluorophores after examining an unstained section for tissue autofluorescence, and keep spectral overlap in mind when assigning channels (standard IF practice). For VPS4B, plan permeabilisation to let antibody reach the cytoplasmic face of vesicle-associated structures, then compare puncta with the vesicle pattern reported by HPA (UniProt O75351: late endosome membrane association and no transmembrane segment; HPA subcellular: vesicles approved). The supplied IF evidence gives no antibody-specific fixation, permeabilisation or dilution setting to transfer into this assay (HPA subcellular: localization summary only).

What should I check when VPS4B DAB staining looks diffuse?

Run a no-primary section to identify signal from the detection system, and examine a nearby section for pigment or tissue deposits before attributing brown color to VPS4B (standard IHC practice). The documented image used biotinylated goat anti-rabbit IgG, a streptavidin-biotin complex and DAB, so include appropriate controls when repeating that detection scheme (caption A03403-1). Block endogenous peroxidase and assess endogenous biotin where relevant to this workflow (standard IHC practice). If background remains broad, compare shorter DAB development and stronger washing while checking that expected cytoplasmic staining is retained (HPA: cytoplasmic tissue expression; standard IHC practice).

How should I score heterogeneous VPS4B staining across sections? ⚠ ANSWER MARKED FOR VERIFICATION

Define the cell population and tissue region before scoring, then apply the same threshold and DAB development conditions to every section (standard IHC practice). An H-score combines the percentage of cells at each intensity and spans 0–300; alternatively report percent positive cells or positive-cell density per mm² (standard IHC practice). Normalize cell counts to the number of eligible cells, and normalize density to analyzable tissue area after excluding folds and necrosis (standard IHC practice). Report epithelial and other cell populations separately when their staining differs, because the reference atlas reports cell-type-specific levels across tissues (HPA: tissue IHC profile).

How can I distinguish true VPS4B staining from an artefact?

Favor reproducible cytoplasmic staining within intact cells, with vesicle-like detail when visible, over isolated nuclear or continuous surface staining (HPA: cytoplasmic tissue expression; HPA subcellular: vesicles approved; UniProt O75351: no transmembrane segment). Check cell identity: HPA reports high staining in esophageal squamous epithelial cells but no detection in lung alveolar cells, while the catalog image shows a human lung cancer section (HPA: tissue IHC; caption A03403-1). A positive tumor section therefore does not establish positivity in adjacent alveolar cells (caption A03403-1; HPA: lung alveolar cells not detected). Exclude edge staining, necrotic regions and signal retained in a no-primary or peroxidase-control section before interpreting DAB as VPS4B (standard IHC practice).

Boster reagents

Best VPS4B / Vacuolar protein sorting-associated protein 4B IHC Antibodies

A03403-1 has real IHC data from paraffin sections of human lung cancer tissue (catalog IHC caption); its listed reactivity is human, mouse, and rat (catalog reactivity).

Real IHC data

Anti-VPS4B/MIG1 Antibody ®

Cat # A03403-1

View product →

A03403-1 is listed for IHC, and its image shows chromogenic detection in a paraffin section of human lung cancer tissue (catalog applications; catalog IHC caption). Human, mouse, and rat are listed as reactive species, but the supplied IHC image documents human tissue only (catalog reactivity; catalog IHC caption).

Which to pick: Choose A03403-1 for human tissue IHC: its caption documents EDTA pH 8 retrieval and 1 μg/ml primary antibody in a paraffin section; the fixative is unreported (catalog IHC caption). No IF/ICC application or image is supplied, so there is no IF/ICC validated SKU to recommend here (catalog applications; catalog IF images). For mouse or rat work, A03403-1 has listed species reactivity, but its supplied IHC evidence is human only; it is a rabbit antibody with no clone reported (catalog reactivity; catalog IHC caption; catalog host; catalog clone).

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

References

UniProt Consortium. UniProt entry O75351 (VPS4B_HUMAN, Vacuolar protein sorting-associated protein 4B).
Human Protein Atlas. VPS4B tissue IHC expression (reliability: Approved).
Human Protein Atlas. VPS4B subcellular location (ICC-IF): Mainly localized to vesicles..
Human Protein Atlas. VPS4B antibody validation summary (3 antibodies).
Dysregulation of the endosomal sorting complex III is linked to neurodegeneration in progressive multiple sclerosis. Brain pathology (Zurich, Switzerland) 2026 — PMC12695678.
Synthetic lethality between VPS4A and VPS4B triggers an inflammatory response in colorectal cancer. EMBO molecular medicine 2020 — PMC7005644.
Vps4b heterozygous mice do not develop tooth defects that replicate human dentin dysplasia I. BMC genetics 2019 — PMC6330468.
Cytoplasmic YAP1-mediated ESCRT-III assembly promotes autophagic cell death and is ubiquitinated by NEDD4L in breast cancer. Cancer communications (London, England) 2023 — PMC10174091.
PubMed PMID:11563910 — UniProt-cited evidence.
PubMed PMID:12594041 — UniProt-cited evidence.
PubMed PMID:9653160 — UniProt-cited evidence.