TRPV4 / Transient receptor potential cation channel subfamily V member 4 · IHC design guide

Design Immunohistochemistry for TRPV4

Plan TRPV4 IHC on paraffin sections using 1–2 μg/mL primary antibody (datasheet A00565). Assess cytoplasmic and membranous staining (HPA tissue IHC), while accounting for the very low consistency between antibody staining and RNA expression (HPA tissue IHC).

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

Immunohistochemistry protocol sheet for TRPV4 (IHC for TRPV4): expected localisation Cytoplasmic and membranous tissue staining (HPA tissue IHC), antibody A00565, validated IHC image, and IHC protocol steps — Printable **TRPV4** IHC protocol sheet — expected localisation Cytoplasmic and membranous tissue staining (HPA tissue IHC), antibody A00565, controls and protocol steps. Open the full TRPV4 IHC guide →

Navigate to: Protocols Staining pattern Controls Antibodies

TRPV4 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	Cytoplasmic and membranous staining in several tissues (HPA tissue IHC)
	Antigen retrieval	Citrate pH 6 HIER, heat-mediated (datasheet A00565)
	Positive control ⓘ	Adrenal gland+4 more · see all
	Negative control ⓘ	Adipose tissue+4 more · see all

Important caveats

Reasons your staining may differ from the expected pattern.

	Fixation	Formaldehyde-fixed paraffin sections (selected-SKU IHC image A00565)
	Caveat	Very low staining–RNA concordance (HPA tissue IHC)
	Regulation	WNK kinases inhibit surface localisation (UniProt)
	Isoform / epitope	Six isoforms; check epitope coverage and membrane side (UniProt)

Section 1

Recommended TRPV4 IHC & IF Protocols

The catalog antibody protocol is accompanied by published chromogenic TRPV4 IHC methods for lung tissue and intervertebral discs (PMC8740047; PMC5072776).

Recommended immunohistochemistry (IHC-P) protocol parameters
Sample	Formaldehyde-fixed, paraffin-embedded human liver tissue (datasheet A00565)
Fixation	Image formalin-fixed; duration unreported (datasheet A00565); verify before use.
Sectioning	4–5 µm sections on charged slides (standard)
Deparaffinisation	Xylene, graded ethanol series to water (standard)
Antigen retrieval	Heat retrieval: Citrate pH 6 (datasheet A00565); 20 min, 95–100 °C (standard)
Peroxidase block	3% H2O2, 10 min, room temperature (standard)
Blocking	10% serum (datasheet A00565)
Primary antibody	Rabbit anti-TRPV4, 1-2 μg/mL (datasheet A00565)
Primary incubation	Overnight at 4 °C (datasheet A00565)
Detection	HRP-polymer secondary, DAB chromogen 5–10 min (standard)
Counterstain	Hematoxylin, blue, dehydrate and mount (standard)
Expected result	TRPV4-positive staining in glandular cells of adrenal gland (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 citrate retrieval at pH 6 for paraffin sections (datasheet A00565; PMC8740047). The disc protocol used deplasticized, methyl methacrylate-embedded sections (PMC5072776).

Section 2

What Is the Expected TRPV4 Staining Pattern?

TRPV4 is a six-pass membrane protein with cytoplasmic termini; UniProt also places it at apical membranes, adherens junctions, cilia and endoplasmic reticulum (UniProt Q9HBA0 topology; subcellular location). In tissue IHC, expect cytoplasmic and membranous staining in selected cell populations, including adrenal glandular cells, pancreatic endocrine cells and cortical neurons (HPA: tissue IHC). Treat these examples as provisional: HPA rates the tissue staining Uncertain because antibody staining and RNA expression show very low consistency (HPA: reliability).

What am I looking at on my slide?
Membranous staining, with some cytoplasmic signal, in selected glandular or neuronal cells.	This fits the reported tissue profile and membrane/ER localization (HPA: cytoplasmic and membranous expression; UniProt Q9HBA0 subcellular location). Interpret the pattern alongside controls because HPA rates tissue IHC Uncertain (HPA: reliability).
Predominantly nuclear staining, with little signal at the membrane or in cytoplasm.	A nuclear-dominant pattern does not fit the listed TRPV4 locations (UniProt Q9HBA0 subcellular location). Review antibody specificity and detection controls before scoring it as positive (standard IHC practice).
Strong signal in a cell population reported as not detected, such as colon glandular cells.	The result conflicts with that HPA tissue example (HPA: colon glandular cells, Not detected). Consider cross-reactivity or endogenous detection activity; HPA's Uncertain rating means the reference pattern is not definitive (HPA: reliability; standard IHC practice).
Similar diffuse staining across cells, stroma and the negative control.	This distribution favors background from the staining or detection workflow over cell-restricted TRPV4 signal (standard IHC practice). Compare the primary-antibody omission control and tissue architecture before assigning localization (standard IHC practice).
No staining in an adrenal gland section expected to show glandular-cell signal.	HPA reports High staining in adrenal glandular cells, so the run needs review (HPA: adrenal gland, glandular cells, High). Check controls and tissue preservation; absence alone cannot establish TRPV4 loss because HPA rates the IHC evidence Uncertain (HPA: reliability; standard IHC practice).

💡Expected TRPV4 appearanceCall a result provisionally positive when selected cells show membranous and/or cytoplasmic signal, potentially prominent in HPA High populations such as pancreatic endocrine cells; uniform diffuse or nuclear-dominant staining is suspect (HPA: tissue IHC, pancreatic endocrine cells High, reliability Uncertain; UniProt Q9HBA0 subcellular location; standard IHC practice).

How each factor affects the staining
Compartment and epitope orientation	TRPV4 has six transmembrane segments, cytoplasmic N- and C-termini, and extracellular loops (UniProt Q9HBA0 topology). Epitope position is not supplied here, so these data cannot specify an antibody-specific retrieval or permeabilisation condition (UniProt Q9HBA0 topology; standard IHC practice).
Intracellular pool	TRPV4 is also listed in the endoplasmic reticulum, where putative tetramers assemble (UniProt Q9HBA0 subcellular location). Cytoplasmic staining can therefore fit the biology, but it requires the same specificity controls as membrane staining (HPA: cytoplasmic and membranous profile; standard IHC practice).
Isoforms	UniProt lists six TRPV4 isoforms and reports isoform-specific self-association (UniProt Q9HBA0 isoforms; subunit). Without an antibody epitope or isoform-reactivity record, a tissue pattern cannot be assigned to one isoform (UniProt Q9HBA0 isoforms; standard IHC practice).
Reference-pattern confidence	HPA labels antibody HPA007150 IHC Uncertain and reports very low agreement between staining and RNA data (HPA: antibody validation; tissue reliability). Use HPA High and Not detected examples as comparison points, not proof of antibody specificity or universal tissue status (HPA: tissue IHC; reliability).

Why is my staining missing, weak or wrong?
Situation	Likely cause	Next action
Little or no chromogenic signal in a comparison tissue.	The staining run may have failed, or the chosen section may lack the reported cell population (standard IHC practice). HPA examples remain provisional (HPA: reliability Uncertain).	Verify the detection controls and morphology; inspect the specific HPA-listed cells, such as adrenal glandular cells, before changing IHC conditions (HPA: adrenal gland, glandular cells High; standard IHC practice).
Predominantly nuclear signal.	The pattern conflicts with listed membrane, junctional, ciliary and ER locations (UniProt Q9HBA0 subcellular location).	Review primary-antibody omission and antibody-specificity controls; score nuclear-only signal cautiously (standard IHC practice; HPA: IHC Uncertain).
Signal appears in colon glandular cells or adipocytes.	HPA lists these cell types as Not detected, though its tissue IHC assessment is Uncertain (HPA: colon glandular cells and adipocytes Not detected; reliability).	Check morphology, antibody specificity and endogenous detection activity before calling the staining TRPV4-positive (standard IHC practice).
Diffuse brown background obscures cell boundaries.	Nonspecific binding or endogenous detection activity can obscure compartment assignment (standard IHC practice).	Compare primary-antibody omission controls; review blocking, washes and detection chemistry using standard IHC controls (standard IHC practice).
Membrane and cytoplasmic staining vary between sections.	Both compartments are reported for tissue staining, while UniProt also lists an ER pool (HPA: tissue profile; UniProt Q9HBA0 subcellular location).	Score each compartment separately in the same named cell type and compare matched controls; avoid treating membrane-only staining as the sole acceptable pattern (HPA: tissue profile; standard IHC practice).
What should an IF/ICC image show?	HPA summarizes TRPV4 as Membrane but provides no ICC-IF image cell lines or main-location detail here (HPA: subcellular summary).	Assess membrane enrichment with compartment and negative controls; use this page's IHC tissue pattern only as context, not an IF/ICC validation claim (HPA: subcellular summary; tissue IHC reliability Uncertain; standard IF practice).

Source: UniProt Q9HBA0 Human Protein Atlas tissue Human Protein Atlas subcellular

Experiment evidence

IHC & IF images showing TRPV4 staining

Immunohistochemistry Validation of TRPV4 in Human Liver Immunohistochemical analysis of paraffin-embedded human liver tissue using anti-TRPV4 antibody (A00565) at 2 μg/ml. Tissue was fixed with formaldehyde and blocked with 10% serum for 1 h at RT; antigen retrieval was by heat mediation with a citrate buffer (pH6). Samples were incubated with primary antibody overnight at 4°C. A goat anti-rabbit IgG H&L (HRP) at 1/250 was used as secondary. Counter stained with Hematoxylin.

A00565 · IHCImmunohistochemistry Validation of TRPV4 in Human Liver Immunohistochemical analysis of paraffin-embedded human liver tissue using anti-TRPV

Immunohistochemistry Validation of TRPV4 in Mouse Kidney Immunohistochemical analysis of paraffin-embedded mouse kidney tissue using anti-TRPV4 antibody (A00565) at 2 μg/ml. Tissue was fixed with formaldehyde and blocked with 10% serum for 1 h at RT; antigen retrieval was by heat mediation with a citrate buffer (pH6). Samples were incubated with primary antibody overnight at 4°C. A goat anti-rabbit IgG H&L (HRP) at 1/250 was used as secondary. Counter stained with Hematoxylin.

A00565 · IHCImmunohistochemistry Validation of TRPV4 in Mouse Kidney Immunohistochemical analysis of paraffin-embedded mouse kidney tissue using anti-TR

A00565 · IFImmunofluorescence of TRPV4 in human kidney tissue with TRPV4 antibody at 5 μg/ml. Red: TRPV4 Antibody (A00565) Blue: DAPI staining

HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000111199-TRPV4/tissue/cerebral+cortex#imid_2317992

HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000111199-TRPV4/tissue/colon#imid_2317934

HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000111199-TRPV4/tissue/liver#imid_2317935

HPA · PancreasHuman Protein Atlas IHC, Pancreas. Source: https://www.proteinatlas.org/ENSG00000111199-TRPV4/tissue/pancreas#imid_2317878

← swipe / scroll for more images →

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

Sample controls for TRPV4 IHC & IF

🧪Run duodenum first: glandular cells should stain (HPA: High in duodenal glandular cells). Use colon glandular cells as the negative tissue (HPA: Not detected in colonic glandular cells); on the duodenum slide, assess non-glandular stromal cells as an internal background reference, without assuming they are TRPV4-negative.

Positive control tissue: Adrenal gland (Glandular cells, HPA High)

Negative control tissue: Adipose tissue (HPA Not detected)

ICC-IF cell lines (HPA subcellular resource): HPA carries no ICC-IF cell line for TRPV4; derive a cell-line control from the positive tissue's cell type (Glandular cells) and confirm it by RNA or western blot first.

Technical controls: Include no-primary (secondary-only) and host- and isotype-matched IgG controls, plus TRPV4-knockout tissue if available (standard IHC practice). In duodenum, check for endogenous peroxidase and, if using avidin–biotin detection, endogenous biotin background (standard IHC practice).

⚠️Feasibility: The A00565 paraffin-section caption reports formaldehyde fixation and heat retrieval in citrate buffer at pH 6, but gives no fixation duration (A00565 tissue-IHC caption). A target-specific fixation window and the effect of omitting retrieval are unreported; the caption alone does not establish an FFPE protocol (A00565 tissue-IHC caption). No matched frozen-section or IF/ICC comparison is supplied, so relative ease is unreported; duodenal endogenous biotin can complicate avidin–biotin detection (standard IHC practice).

HPA tissue IHC evidence for TRPV4

Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Uncertain — Very low consistency between antibody staining and RNA expression data. Pending external verification.). Rows are taken directly from the HPA tissue chart — click any row's HPA link to view the source.

Positive expression · recommended positive controls

Tissue	Cell type	Level	Evidence	Source
Adrenal gland	Glandular cells	High	Protein (IHC)	HPA →
Cerebral cortex	Neuronal cells	High	Protein (IHC)	HPA →
Duodenum	Glandular cells	High	Protein (IHC)	HPA →
Hippocampus	Neuronal cells	High	Protein (IHC)	HPA →
Pancreas	Pancreatic endocrine cells	High	Protein (IHC)	HPA →

Undetected expression · recommended negative controls

Tissue	Cell type	Level	Evidence	Source
Adipose tissue	Adipocytes	Not detected	Protein (IHC)	HPA →
Bone marrow	Hematopoietic cells	Not detected	Protein (IHC)	HPA →
Cervix	Glandular cells	Not detected	Protein (IHC)	HPA →
Colon	Glandular cells	Not detected	Protein (IHC)	HPA →
Epididymis	Glandular cells	Not detected	Protein (IHC)	HPA →

Section 3

Advanced TRPV4 IHC Tips

Troubleshoot TRPV4 staining in paraffin sections by checking retrieval, tissue controls, cellular localisation and the limits of the available validation evidence.

How should I retrieve TRPV4 antigen when paraffin sections stain weakly?

Start with heat-mediated antigen retrieval in citrate buffer at pH 6 (datasheet A00565). The selected paraffin-section image used this retrieval before overnight primary incubation at 4°C (datasheet A00565); use it as the reference condition when comparing runs. If staining remains weak, check whether sections were fully deparaffinised and whether heating and cooling were consistent across slides (standard IHC practice). Change retrieval conditions only in a controlled comparison with matched positive and negative controls, because stronger staining alone cannot establish TRPV4 specificity, especially given the uncertain consistency of tissue staining with RNA data (HPA: reliability Uncertain).

Could fixation explain weak or uneven TRPV4 staining in my sections?

Target-specific sensitivity of TRPV4 IHC to fixation is unknown from the supplied evidence; do not infer it from tissue staining patterns or channel topology (supplied evidence; UniProt Q9HBA0 topology). Record fixative, fixation duration, section age and processing history for every comparison, then test matched sections under the same retrieval and detection conditions (standard IHC practice). Uneven staining confined to poorly preserved or thick areas should prompt a section-quality check before increasing primary antibody concentration (standard IHC practice). Compare morphology and a matched control section alongside the TRPV4 slide, since changing fixation and retrieval together prevents a clear diagnosis of weak staining (standard IHC practice).

Which cellular staining pattern should I expect for TRPV4 in chromogenic IHC?

Assess membrane-associated staining and plausible cytoplasmic signal: TRPV4 is annotated at the cell membrane, apical membrane, adherens junctions, cilium and endoplasmic reticulum (UniProt Q9HBA0 subcellular location). The tissue IHC profile describes cytoplasmic and membranous expression, but its reliability is uncertain (HPA: tissue IHC profile; HPA: reliability Uncertain). Evaluate localisation within identified cells rather than calling a whole tissue positive from diffuse colour, and compare equivalent compartments on adjacent sections (standard IHC practice). A purely nuclear pattern lacks support from the supplied localisation record and merits review of counterstain, background and antibody specificity before biological interpretation (UniProt Q9HBA0 subcellular location; standard IHC practice).

Can this antibody distinguish TRPV4 isoforms or reveal an inaccessible epitope?

Do not assign isoform specificity without an epitope map or isoform-specific validation for the catalog antibody (supplied evidence). TRPV4 has 6 annotated isoforms, while its reference topology places residues 1–469 and 723–871 on the cytoplasmic side and several short loops extracellularly (UniProt Q9HBA0 isoforms and topology). An antibody to a cytoplasmic epitope may depend on section processing for access, but the epitope side for A00565 is not supplied (UniProt Q9HBA0 topology; supplied evidence). If staining differs among specimens, first compare retrieval and preservation, then seek epitope documentation or independent validation before attributing that difference to an isoform (standard IHC practice).

How can I assess TRPV4 by multiplex IF alongside this IHC workflow?

Treat IF as a separate assay and establish its own fixation, permeabilisation and antibody controls rather than transferring performance from the paraffin IHC image (standard IF practice; datasheet A00565). Multiplex TRPV4 with a validated marker for the expected cell type, such as a neuronal marker when examining neuronal cells, which HPA scores high in cerebral cortex and hippocampus (HPA: cerebral cortex and hippocampus neuronal cells High; standard IF practice). Choose fluorophores after measuring tissue autofluorescence in each channel and include single-stain controls for spectral overlap (standard IF practice). Set permeabilisation according to a documented epitope: cytoplasmic regions require access across membranes, whereas extracellular loops may be assessed without permeabilisation; A00565's epitope is unspecified (UniProt Q9HBA0 topology; supplied evidence; standard IF practice).

What should I check when TRPV4 chromogenic staining appears diffuse?

First compare a no-primary control and a matched tissue control to separate detection-system colour from primary-dependent staining (standard IHC practice). In the selected paraffin-section example, sections were blocked with 10% serum for 1 hour at room temperature, then received 2 μg/ml primary overnight at 4°C (datasheet A00565). Check peroxidase blocking, secondary-antibody compatibility, washes and chromogen development as general chromogenic IHC variables (standard IHC practice). If colour remains diffuse, inspect tissue edges, folds and damaged regions separately from intact cells; raising the primary concentration before resolving those patterns can obscure interpretable membrane or cytoplasmic staining (standard IHC practice; UniProt Q9HBA0 subcellular location).

How should I score TRPV4 staining across paraffin-section specimens? ⚠ ANSWER MARKED FOR VERIFICATION

Define the cell population and anatomical compartment before scoring, because TRPV4 has several annotated cellular locations and HPA reports cytoplasmic and membranous tissue staining (UniProt Q9HBA0 subcellular location; HPA: tissue IHC profile). For comparable sections, record the percentage of positive cells and an intensity-weighted H-score, with intensity categories defined before review (standard IHC practice). Normalise positive-cell counts to the total eligible cells of the same type, or report positive-cell density per mm² of viable, evaluable tissue (standard IHC practice). Apply one threshold and imaging setup across groups, exclude damaged areas, and report control results because the HPA staining profile has uncertain reliability (standard IHC practice; HPA: reliability Uncertain).

When is a positive TRPV4 IHC result biologically credible?

A credible result should occupy identified cells in a plausible membrane-associated or cytoplasmic compartment and reproduce across comparable sections (UniProt Q9HBA0 subcellular location; HPA: tissue IHC profile; standard IHC practice). For context, HPA scores neuronal cells in cerebral cortex and hippocampus High, while adipocytes in adipose tissue are Not detected; those observations are provisional because overall reliability is Uncertain (HPA: tissue IHC profile and reliability). Treat nuclear-only colour, staining concentrated at cut edges or necrotic areas, and colour persisting in a no-primary control as possible artefacts (UniProt Q9HBA0 subcellular location; standard IHC practice). Resolve such patterns with morphology, matched controls and independent target validation before claiming a tissue-specific TRPV4 difference (standard IHC practice; HPA: reliability Uncertain).

Boster reagents

Best TRPV4 / Transient receptor potential cation channel subfamily V member 4 IHC Antibodies

The IHC-validated antibody has paraffin-section images from human liver, mouse kidney and rat kidney, plus an IF image from human kidney (catalog IHC and IF captions).

Real IHC data

Anti-TRPV4 Antibody

Cat # A00565

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A00565 is listed for IHC-P and IF in human, mouse and rat samples (catalog applications and reactivity). Its IHC captions show human liver, mouse kidney and rat kidney paraffin sections; its IF caption shows human kidney tissue (catalog image captions).

Which to pick: Choose A00565 for tissue IHC: its own captions document formaldehyde-fixed paraffin sections with citrate pH 6 heat retrieval and 2 μg/mL primary antibody (A00565 IHC captions). Choose A00565 for tissue IF, with human kidney imaged at 5 μg/mL; ICC validation is unreported (A00565 IF caption; catalog applications). For cross-species IHC, A00565 has human, mouse and rat paraffin-section images and is listed as polyclonal (A00565 IHC captions; catalog dilution_raw).

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

References

UniProt Consortium. UniProt entry Q9HBA0 (TRPV4_HUMAN, Transient receptor potential cation channel subfamily V member 4).
Human Protein Atlas. TRPV4 tissue IHC expression (reliability: Uncertain).
Human Protein Atlas. TRPV4 subcellular location (ICC-IF): Membrane.
Human Protein Atlas. TRPV4 antibody validation summary (1 antibodies).
TRPV4 plays an important role in rat prefrontal cortex changes induced by acute hypoxic exercise. Saudi journal of biological sciences 2019 — PMC6734159.
Transient Receptor Potential Cation Channel Subfamily V Member 4 Mediates Pyroptosis in Chronic Obstructive Pulmonary Disease. Frontiers in physiology 2021 — PMC8740047.
Reduced tissue osmolarity increases TRPV4 expression and pro-inflammatory cytokines in intervertebral disc cells. European cells & materials 2016 — PMC5072776.
Transient receptor potential vanilloid 4 channels contribute to the initiation of water-induced swallowing reflexes. Research square 2026 — PMC13119302.
PubMed PMID:11081638 — UniProt-cited evidence.
PubMed PMID:11025659 — UniProt-cited evidence.
PubMed PMID:12692122 — UniProt-cited evidence.