SPRY4 / Protein sprouty homolog 4 · IHC design guide

Design Immunohistochemistry for SPRY4

Plan SPRY4 staining in paraffin sections using its reported cytoplasmic tissue pattern and cell-level controls (HPA tissue IHC). The guide also covers fixation consistency, antigen retrieval, and interpretation of possible EGF-linked redistribution (UniProt).

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

Immunohistochemistry protocol sheet for SPRY4 (IHC for SPRY4): expected localisation Predominantly cytoplasmic tissue staining (HPA tissue IHC), antibody A04343-2, validated IHC image, and IHC protocol steps — Printable **SPRY4** IHC protocol sheet — expected localisation Predominantly cytoplasmic tissue staining (HPA tissue IHC), antibody A04343-2, controls and protocol steps. Open the full SPRY4 IHC guide →

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SPRY4 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	Predominantly cytoplasmic tissue staining (HPA tissue IHC)
	Staining pattern	Cytoplasmic staining in respiratory epithelium, tubules and hepatocytes (HPA tissue IHC)
	Antigen retrieval	EDTA pH 8.0 HIER, heat-mediated (datasheet A04343-2)
	Positive control ⓘ	Bronchus+4 more · see all
	Negative control ⓘ	Bone marrow

Important caveats

Reasons your staining may differ from the expected pattern.

	Fixation	Keep fixation conditions consistent across sections. (standard IHC practice; not target-specific)
	Caveat	EGF may shift SPRY4 toward membrane ruffles (UniProt)
	Regulation	EGF-linked redistribution, by similarity (UniProt)
	Isoform / epitope	Two isoforms, A and C; epitope coverage is unspecified (UniProt)

Section 1

Recommended SPRY4 IHC & IF Protocols

The catalog antibody’s IHC-P protocol is paired with three published SPRY4 IHC protocols (datasheet: A04343-2; PMC8521147; PMC9208480; PMC6475890).

Recommended immunohistochemistry (IHC-P) protocol parameters
Sample	Paraffin-embedded human liver cancer tissue; fixative not specified (datasheet A04343-2)
Fixation	Image fixative and duration unreported (datasheet A04343-2); 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 A04343-2); 20 min, 95–100 °C (standard)
Peroxidase block	3% H2O2, 10 min, room temperature (standard)
Blocking	10% goat serum (datasheet A04343-2)
Primary antibody	Rabbit anti-SPRY4, 0.5-1μg/ml (datasheet A04343-2)
Primary incubation	Overnight at 4 °C (datasheet A04343-2)
Detection	Streptavidin-biotin complex (SABC), DAB chromogen (datasheet A04343-2)
Counterstain	Hematoxylin, blue, dehydrate and mount (standard)
Expected result	SPRY4-positive staining in respiratory epithelial cells of bronchus (HPA tissue IHC: High). HPA tissue profile: General cytoplasmic expression. No signal in the no-primary control.

💡Decision noteStart with heat-mediated EDTA pH 8.0 retrieval for the catalog antibody (datasheet: A04343-2); the cited excerpts do not specify a retrieval buffer (PMC8521147; PMC9208480; PMC6475890).

Section 2

What Is the Expected SPRY4 Staining Pattern?

SPRY4 should show predominantly cytoplasmic staining in paraffin section IHC, with strong signal in several epithelial and glandular cell populations (HPA tissue IHC: general cytoplasmic expression; High in specified cell types). HPA rates the tissue staining profile Enhanced, with medium consistency against RNA data (HPA tissue IHC). UniProt places SPRY4 in the cytoplasm and reports no transmembrane segment (UniProt Q9C004 topology and subcellular location).

What am I looking at on my slide?
Cytoplasmic signal in bronchial respiratory epithelium, kidney tubules, or hepatocytes.	This matches cell populations scored High by HPA (HPA tissue IHC). Judge the signal within the named cells, since neighboring cells in the same section need not share their staining level.
Strong nuclear-only or sharply surface-confined staining, with little cytoplasmic signal.	Treat this as a compartment mismatch requiring validation: HPA describes general cytoplasmic tissue staining (HPA tissue IHC). UniProt reports cytoplasm and EGF-associated ruffle localization by similarity, but no transmembrane segment (UniProt Q9C004).
Strong staining centered on cells other than the HPA-listed positive population.	Check cell identity before calling SPRY4 positive. In an unexpected population, cross-reactivity or endogenous detection activity is possible (general IHC practice); HPA tissue scores apply to named cell types, not every cell in an organ (HPA tissue IHC).
Diffuse color across tissue, stroma, and blank spaces, without cell boundaries.	This is more consistent with background than an interpretable SPRY4 pattern (general IHC practice). HPA reports a cellular, generally cytoplasmic profile (HPA tissue IHC); inspect controls before assigning a positive score.
No cytoplasmic signal in an adequately preserved HPA High cell population.	Investigate assay performance before inferring absence of SPRY4 (general IHC practice). Bronchial respiratory epithelial cells, kidney tubular cells, and hepatocytes are HPA High examples (HPA tissue IHC), though staining depends on the tested assay.

💡Expected SPRY4 appearanceCall a convincing positive when the named HPA High cells show clear cytoplasmic staining; isolated nuclear signal or diffuse tissue-wide color is suspect (HPA tissue IHC; general IHC practice).

How each factor affects the staining
Choice of tissue and cell population	HPA scores bronchial respiratory epithelium, duodenal and small-intestinal glandular cells, kidney tubules, hepatocytes, lung type II alveolar cells, and lymph-node non-germinal-center cells High; stomach glandular cells are Medium (HPA tissue IHC).
Low and undetected comparisons	HPA reports low staining in several populations, including cerebral-cortex endothelium and smooth muscle, and does not detect SPRY4 in bone-marrow hematopoietic cells (HPA tissue IHC). Use these as context, not as absolute assay-negative controls.
Antibody validation	The tissue profile is Enhanced with medium antibody–RNA consistency (HPA tissue IHC). Among listed antibodies, CAB008086 has Enhanced IHC validation; HPA055471 and HPA072661 have Approved ICC status but no supplied IHC status (HPA antibodies).
Subcellular interpretation	UniProt reports cytoplasm at baseline and EGF-associated movement to membrane ruffles by similarity (UniProt Q9C004). This does not establish that every paraffin section should show visible ruffle staining; HPA describes general cytoplasmic tissue IHC (HPA tissue IHC).
IF/ICC Q&A: what localization should be expected?	HPA reports mainly Golgi localization, with additional cytosol, in ICC-IF (HPA subcellular). Interpret that as an ICC-IF observation; the supplied tissue IHC summary supports general cytoplasmic staining, without resolving a Golgi pattern in sections (HPA tissue IHC).
Isoforms and antigen retrieval	UniProt lists isoforms A and C (UniProt Q9C004). Epitope coverage and a SPRY4-specific retrieval condition are not supplied, so neither isoform recognition nor retrieval sensitivity can be predicted from these records.

Why is my staining missing, weak or wrong?
Situation	Likely cause	Next action
Expected HPA High cells are unstained.	Possible detection failure, unsuitable assay settings, or sample variation (general IHC practice); HPA High is an observed profile, not a guarantee for every section (HPA tissue IHC).	Check a same-run positive control in a named HPA High cell type, then review the IHC-validated antibody’s documented conditions and detection controls (HPA tissue IHC; general IHC practice).
Only nuclei stain strongly.	The compartment differs from HPA’s general cytoplasmic tissue pattern (HPA tissue IHC); nonspecific binding or detection artifact is possible (general IHC practice).	Review cell boundaries and counterstain, compare an appropriate control, and reassess the antibody and detection settings before scoring nuclei as SPRY4 (general IHC practice).
Unexpected cells stain more strongly than the named positive cells.	Cell misidentification, cross-reactivity, or endogenous detection activity may explain the result (general IHC practice); HPA scores specific cell populations (HPA tissue IHC).	Confirm morphology and location, then use suitable reagent and endogenous-activity controls for the chromogenic detection system (general IHC practice).
Color is diffuse or present outside cells.	Excess background can obscure the cytoplasmic pattern expected in tissue IHC (HPA tissue IHC; general IHC practice).	Inspect negative detection controls and optimize blocking, washing, antibody concentration, and chromogen development using the assay’s documented conditions (general IHC practice).
Bone-marrow hematopoietic cells show strong staining.	HPA reports these cells as Not detected (HPA tissue IHC), so prominent signal warrants a check for nonspecific or endogenous detection (general IHC practice).	Compare with a same-run HPA High cell population and the appropriate detection controls; do not use the bone-marrow observation alone to redefine SPRY4 localization (HPA tissue IHC; general IHC practice).
A punctate perinuclear signal is difficult to score in a tissue section.	HPA ICC-IF reports mainly Golgi localization, whereas its tissue IHC summary says general cytoplasmic expression (HPA subcellular; HPA tissue IHC).	Score the identifiable tissue cell population and overall cytoplasmic signal first; avoid requiring a resolvable Golgi pattern for a positive chromogenic IHC call (HPA tissue IHC; general IHC practice).

Source: UniProt Q9C004 Human Protein Atlas tissue Human Protein Atlas subcellular

Experiment evidence

IHC & IF images showing SPRY4 staining

IHC analysis of Sprouty 4/Spry-4/SPRY4 using anti-Sprouty 4/Spry-4/SPRY4 antibody (A04343-2). Sprouty 4/Spry-4/SPRY4 was detected in paraffin-embedded section of human liver 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-Sprouty 4/Spry-4/SPRY4 Antibody (A04343-2) 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.

A04343-2 · IHCIHC analysis of Sprouty 4/Spry-4/SPRY4 using anti-Sprouty 4/Spry-4/SPRY4 antibody (A04343-2). Sprouty 4/Spry-4/SPRY4 was detected in paraffi

HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000187678-SPRY4/tissue/cerebral+cortex#imid_2262780

HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000187678-SPRY4/tissue/colon#imid_2262706

HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000187678-SPRY4/tissue/liver#imid_2262723

HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000187678-SPRY4/tissue/kidney#imid_2262724

HPA · ICC-IF PC-3Human Protein Atlas immunofluorescence, PC-3, antibody HPA055471; green = SPRY4, blue = nucleus, red = microtubules.

← swipe / scroll for more images →

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

Sample controls for SPRY4 IHC & IF

🧪Run liver first: hepatocytes should stain (HPA: High in hepatocytes), and the selected-SKU caption documents staining in a paraffin section of human liver cancer tissue (caption: A04343-2). Run bone marrow as the negative tissue (HPA: Not detected in hematopoietic cells); on the liver slide, compare hepatocyte staining with background in other tissue elements, without assuming those elements are SPRY4-negative (HPA: High in hepatocytes).

Positive control tissue: Bronchus (Respiratory epithelial cells, HPA High)

Negative control tissue: Bone marrow (HPA Not detected)

ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show SPRY4 in PC-3, U-251MG, U2OS, SK-MEL-30, with annotated localisation: Golgi apparatus (approved) (HPA subcellular).

Technical controls: Include no-primary (secondary-only) and concentration-matched rabbit immunoglobulin controls appropriate to the primary antibody’s clonality (caption: rabbit primary antibody), plus a SPRY4-knockout biological control where available (standard IHC practice). For liver, block endogenous peroxidase and endogenous biotin before the biotin-based DAB detection used in the caption (caption: biotinylated secondary, streptavidin–biotin complex and DAB; standard IHC practice).

⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the exact A04343-2 tissue-IHC caption does not state the fixative (caption: paraffin section; fixative unreported). Heat retrieval with EDTA at pH 8.0 is documented for that section, but its necessity relative to other retrieval conditions is unreported (caption: EDTA heat retrieval); the supplied evidence does not establish whether frozen sections or IF would be easier. Liver endogenous biotin and peroxidase can complicate the caption’s biotin-based chromogenic detection, so assess these sources of background with the technical controls (caption: streptavidin–biotin complex and DAB; standard IHC practice).

HPA tissue IHC evidence for SPRY4

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
Bronchus	Respiratory epithelial cells	High	Protein (IHC)	HPA →
Duodenum	Glandular cells	High	Protein (IHC)	HPA →
Kidney	Cells in tubules	High	Protein (IHC)	HPA →
Liver	Hepatocytes	High	Protein (IHC)	HPA →
Lung	Alveolar cells type II	High	Protein (IHC)	HPA →

Undetected expression · recommended negative controls

Tissue	Cell type	Level	Evidence	Source
Bone marrow	Hematopoietic cells	Not detected	Protein (IHC)	HPA →

Section 3

Advanced SPRY4 IHC Tips

Troubleshoot SPRY4 staining in paraffin section IHC using the catalog antibody’s documented conditions, tissue patterns and expected cellular localisation.

Which retrieval conditions should I try first for weak SPRY4 staining?

Start with heat mediated antigen retrieval in EDTA, pH 8.0, for paraffin sections (datasheet A04343-2). The catalog antibody’s tissue image used that condition before incubation at 1 μg/ml overnight at 4°C (datasheet A04343-2). If staining is weak, compare a longer or shorter heating time on matched sections while keeping the buffer and antibody concentration fixed (standard IHC practice). Include a section expected to stain, such as liver hepatocytes, and assess its cytoplasmic signal alongside the study sample (HPA: High in hepatocytes; HPA: general cytoplasmic expression). Record retrieval time and tissue preservation so stronger DAB does not conceal heat damage (standard IHC practice).

Could fixation explain variable SPRY4 staining between paraffin blocks?

Target specific SPRY4 sensitivity to fixative or fixation duration is unknown from the supplied evidence; the catalog tissue caption does not state its fixative (datasheet A04343-2). Compare matched material fixed for 6 h and 24 h as an experimental check, then process, retrieve and stain both together (standard IHC practice). Keep EDTA, pH 8.0, antibody concentration and detection timing constant so fixation is the planned variable (datasheet A04343-2; standard IHC practice). Review tissue morphology and cytoplasmic staining in an expected positive compartment before attributing a difference to SPRY4 epitope loss (HPA: general cytoplasmic expression; standard IHC practice). Do not infer fixation tolerance from tissue staining patterns or protein topology (HPA: tissue IHC; UniProt Q9C004: no transmembrane segment).

Where should SPRY4 staining appear within positive cells?

Expect a predominantly cytoplasmic pattern when interpreting paraffin section IHC, while allowing focal membrane ruffle signal in an appropriate stimulated context (HPA: general cytoplasmic expression; UniProt Q9C004: EGF associated ruffle translocation by similarity). SPRY4 has no transmembrane segment, so a crisp continuous plasma membrane outline alone warrants scrutiny (UniProt Q9C004: topology; standard IHC practice). Golgi localisation is reported in cell based imaging, with additional cytosol signal, but that result need not resolve into a distinct Golgi pattern in chromogenic tissue sections (HPA: subcellular localisation; standard IHC practice). At 20× and 40×, compare staining within cells against morphology and the no primary control (standard IHC practice). Score cell identity and compartment separately before interpreting focal staining (standard IHC practice).

Can this antibody distinguish SPRY4 isoforms or phosphorylation states in tissue?

SPRY4 has 2 annotated isoforms, A and C, and its SPR domain spans residues 166–273 (UniProt Q9C004). The supplied catalog tissue caption gives staining conditions but no mapped antibody epitope, so isoform coverage and epitope accessibility cannot be assigned (datasheet A04343-2). Phosphoserine at residue 125 is annotated, but ordinary chromogenic signal cannot establish its phosphorylation state without a validated site specific reagent (UniProt Q9C004; standard IHC practice). If staining differs between samples, test specificity with an independent, mapped antibody or an appropriate loss of SPRY4 control under the same retrieval conditions (standard IHC practice). Report signal as SPRY4 immunoreactivity, not an isoform or phosphoform measurement (standard IHC practice).

How should I assess SPRY4 localisation in a multiplex IF experiment?

On the separate IF/ICC guide, pair SPRY4 with a marker for the expected cell type, such as an epithelial marker when examining respiratory epithelium (HPA: High in bronchial respiratory epithelial cells; standard IF practice). Choose a fluorophore channel with low tissue autofluorescence and inspect an unstained section in every channel before interpreting puncta (standard IF practice). Because SPRY4 lacks a transmembrane segment and is reported in cytosol and Golgi, use permeabilisation appropriate for intracellular epitopes when the antibody’s epitope is accessible there (UniProt Q9C004: topology; HPA: subcellular localisation; standard IF practice). Compare mild and stronger permeabilisation on matched samples, keeping exposure fixed, since the catalog’s 1 μg/ml condition documents chromogenic tissue IHC only (datasheet A04343-2; standard IF practice). Confirm apparent colocalisation at single cell resolution (standard IF practice).

How can I separate SPRY4 signal from chromogenic background?

Run a no primary control beside a known staining section and compare DAB deposition at matched development times (standard IHC practice). The catalog tissue image used 10% goat serum blocking, a biotinylated secondary, a streptavidin–biotin complex and DAB (datasheet A04343-2). For that detection scheme, include peroxidase blocking and assess endogenous biotin as potential sources of signal unrelated to antibody binding (standard IHC practice). If diffuse colour persists, shorten DAB development or titrate the primary below the documented 1 μg/ml while checking whether expected hepatocyte cytoplasmic staining remains detectable (datasheet A04343-2; HPA: High in hepatocytes; standard IHC practice). Record control and sample images with matched settings (standard IHC practice).

What scoring method makes SPRY4 IHC comparable across sections? ⚠ ANSWER MARKED FOR VERIFICATION

Define the cell population and cytoplasmic compartment before scoring, because SPRY4 tissue IHC is described as generally cytoplasmic (HPA: general cytoplasmic expression; standard IHC practice). For each section, record the percentage of positive target cells and an H-score using 0–3 intensity categories, then apply the same thresholds across batches (standard IHC practice). Alternatively, report positive cell density per mm² within a prespecified tissue region when cell counts are the outcome (standard IHC practice). Normalise to viable target cells or measured tissue area, excluding necrosis, folds and empty spaces by a written rule (standard IHC practice). Include matched controls and fixed DAB development time because batch variation can shift apparent intensity (standard IHC practice).

When should apparent SPRY4 positivity be treated as artefact?

Treat staining as more credible when it appears in intact cells with the expected cytoplasmic distribution and in a plausible positive cell population, such as hepatocytes (HPA: general cytoplasmic expression; HPA: High in hepatocytes). Strong isolated nuclear staining or a uniform sharp membrane rim needs additional validation against SPRY4’s reported cytoplasmic localisation and lack of a transmembrane segment (HPA: tissue IHC; UniProt Q9C004: topology; standard IHC practice). Inspect section edges and necrotic regions for staining that disregards cell boundaries, and compare these areas with a no primary control (standard IHC practice). Residual endogenous peroxidase can produce DAB colour, so confirm the peroxidase block worked before scoring (standard IHC practice). Review at 20× and 40× with morphology visible (standard IHC practice).

Boster reagents

Best SPRY4 / Protein sprouty homolog 4 IHC Antibodies

A04343-2 has human paraffin-section IHC images (catalog IHC captions). M04343 lists human, mouse, and rat ICC/IF use; no IF image is supplied (catalog applications/reactivity/image alts).

Real IHC data

Anti-Sprouty 4/Spry-4/SPRY4 Antibody ®

Cat # A04343-2

View product →

A04343-2 is listed for human IHC, with images from paraffin-embedded human liver and rectal cancer tissue (catalog applications/reactivity/IHC captions). M04343 is listed for human, mouse, and rat ICC/IF, with no IHC or IF image supplied (catalog applications/reactivity/image alts).

Which to pick: Choose A04343-2 for chromogenic IHC on paraffin sections: its own human liver cancer caption documents EDTA retrieval at pH 8.0 and DAB detection (A04343-2 IHC caption). Its fixative is unreported (A04343-2 IHC caption). Choose M04343 for IF/ICC or mouse and rat samples because it is a rabbit monoclonal, clone 18S63, listed for human, mouse, and rat ICC/IF (M04343 catalog clone/applications/reactivity).

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

References

UniProt Consortium. UniProt entry Q9C004 (SPY4_HUMAN, Protein sprouty homolog 4).
Human Protein Atlas. SPRY4 tissue IHC expression (reliability: Enhanced).
Human Protein Atlas. SPRY4 subcellular location (ICC-IF): Mainly localized to the Golgi apparatus. In addition localized to the cytosol..
Human Protein Atlas. SPRY4 antibody validation summary (3 antibodies).
SPRY4-AS1, A Novel Enhancer RNA, Is a Potential Novel Prognostic Biomarker and Therapeutic Target for Hepatocellular Carcinoma. Frontiers in oncology 2021 — PMC8521147.
Autoregulatory loop between TGF-β1/miR-411-5p/SPRY4 and MAPK pathway in rhabdomyosarcoma modulates proliferation and differentiation. Cell death & disease 2015 — PMC4558514.
Downregulated miR-18a and miR-92a synergistically suppress non-small cell lung cancer via targeting Sprouty 4. Bioengineered 2022 — PMC9208480.
Oncogenic microRNA-411 promotes lung carcinogenesis by directly targeting suppressor genes SPRY4 and TXNIP. Oncogene 2019 — PMC6475890.
PubMed PMID:12027893 — UniProt-cited evidence.
PubMed PMID:14977631 — UniProt-cited evidence.
PubMed PMID:14702039 — UniProt-cited evidence.