SPRED2 / Sprouty-related, EVH1 domain-containing protein 2 · IHC design guide

Design Immunohistochemistry for SPRED2

Plan SPRED2 IHC in paraffin sections using the variable cytoplasmic tissue pattern as a reference (HPA tissue IHC). The catalog antibody has a human liver IHC example and a 2–5 μg/ml IHC dilution range (datasheet A06368-1).

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

Immunohistochemistry protocol sheet for SPRED2 (IHC for SPRED2): expected localisation Cytoplasmic staining is observed in tissue (HPA tissue IHC), antibody A06368-1, validated IHC image, and IHC protocol steps — Printable **SPRED2** IHC protocol sheet — expected localisation Cytoplasmic staining is observed in tissue (HPA tissue IHC), antibody A06368-1, controls and protocol steps. Open the full SPRED2 IHC guide →

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SPRED2 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 is observed in tissue (HPA tissue IHC)
	Staining pattern	Variable cytoplasmic staining across tissue cell types (HPA tissue IHC)
	Antigen retrieval	EDTA pH 8.0 HIER, heat-mediated (datasheet A06368-1)
	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 conditions consistent across sections. (standard IHC practice; not target-specific)
	Caveat	Staining and RNA show medium consistency (HPA tissue IHC)
	Regulation	Expression regulator not specified (UniProt)
	Isoform / epitope	2 isoforms; epitope differences unspecified (UniProt)

Section 1

Recommended SPRED2 IHC & IF Protocols

The catalog antibody uses EDTA pH 8.0 retrieval (datasheet A06368-1). Three published SPRED2 IHC protocols below use citric acid retrieval (PMC8612556; PMC10003366; PMC12563662).

Recommended immunohistochemistry (IHC-P) protocol parameters
Sample	Paraffin-embedded human liver tissue; fixative not specified (datasheet A06368-1)
Fixation	Image fixative and duration unreported (datasheet A06368-1); 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 A06368-1); 20 min, 95–100 °C (standard)
Peroxidase block	3% H2O2, 10 min, room temperature (standard)
Blocking	10% goat serum (datasheet A06368-1)
Primary antibody	Rabbit anti-SPRED2, 2-5 μg/ml (datasheet A06368-1)
Primary incubation	Overnight at 4 °C (datasheet A06368-1)
Detection	HRP-conjugated secondary, DAB chromogen (datasheet A06368-1)
Counterstain	Hematoxylin, blue, dehydrate and mount (standard)
Expected result	SPRED2-positive staining in glandular cells of adrenal gland (HPA tissue IHC: High). HPA tissue profile: General cytoplasmic expression of varying levels. No signal in the no-primary control.

💡Decision noteStart with EDTA pH 8.0 for the catalog antibody (datasheet A06368-1); evaluate citric acid retrieval when adapting a published method (PMC8612556; PMC10003366; PMC12563662).

Section 2

What Is the Expected SPRED2 Staining Pattern?

In paraffin sections, expect predominantly cytoplasmic SPRED2 staining, potentially including vesicle-associated and membrane-adjacent signal (UniProt Q7Z698 localisation; no transmembrane segment). HPA reports general cytoplasmic expression of varying levels, with high staining in several glandular cell types, cardiomyocytes and kidney tubule cells (HPA: tissue IHC). Its tissue IHC assessment is Approved, with medium consistency between antibody staining and RNA expression (HPA: reliability).

What am I looking at on my slide?
Cytoplasmic chromogen in adrenal or salivary glandular cells, cardiomyocytes, or kidney tubule cells.	This matches the reported compartment and examples of high-staining cells (HPA: tissue IHC). Compare the cells with their neighbors and score both the proportion stained and intensity; a positive result does not require every cell to stain equally, because HPA describes varying cytoplasmic levels (HPA: tissue IHC).
Predominantly nuclear chromogen in a paraffin section, with little convincing cytoplasmic staining.	Treat this as a compartment mismatch requiring investigation, rather than confirmed SPRED2 staining: HPA tissue IHC describes general cytoplasmic expression (HPA: tissue IHC). HPA reports uncertain nucleoplasmic localisation in ICC-IF, alongside supported cytosol localisation; that separate observation does not establish a nuclear IHC pattern (HPA: subcellular ICC-IF).
Strong staining chiefly in cells outside the expected positive cell population.	Check cell identity and the detection control before assigning a biological pattern. HPA identifies specific high-staining populations, including pancreatic exocrine glandular cells and kidney tubule cells (HPA: tissue IHC). Unanticipated staining may reflect nonspecific binding or endogenous detection activity (standard IHC practice); the HPA list alone cannot prove cross-reactivity.
Uniform haze across cells and surrounding tissue, obscuring cytoplasmic boundaries.	This is background until a cell-associated pattern can be resolved (standard IHC practice). Compare an antibody-omission control and review blocking, washes, detection reagent and chromogen development (standard IHC practice). A broadly expressed target can complicate visual comparison, since HPA reports low tissue specificity rather than a reliably negative tissue (HPA: RNA specificity).
No convincing stain in an expected high-staining control section.	First check section integrity and the staining run, including retrieval, antibody incubation and detection controls (standard IHC practice). HPA reports high staining in duodenal glandular cells and kidney tubule cells, among other populations (HPA: tissue IHC). A blank control section warrants troubleshooting but does not, by itself, establish a target-specific fixation effect.

💡Expected SPRED2 appearanceCall a result positive when identifiable expected cells show predominantly cytoplasmic staining of discernible intensity; isolated nuclear staining or featureless haze is suspect (HPA: tissue IHC; standard IHC practice).

How each factor affects the staining
Tissue and cell selection	HPA reports high staining in adrenal, duodenal, gallbladder, rectal and salivary glandular cells, cardiomyocytes, kidney tubule cells, and pancreatic exocrine glandular cells (HPA: tissue IHC). Select an identifiable reported population for a positive comparison; HPA supplies no negative tissue list (HPA: tissue IHC).
Compartment and topology	Cytoplasm, secretory vesicle membrane and cell membrane are UniProt localisations; SPRED2 has no transmembrane segment (UniProt Q7Z698 localisation and topology). Interpret a membrane-adjacent accent within a broader cellular pattern cautiously: this record gives no IHC criterion requiring a crisp membrane outline.
Evidence strength	The HPA tissue IHC assessment is Approved but has medium consistency with RNA expression (HPA: reliability). HPA064394 is IHC Approved; the supplied record gives no IHC status for HPA038256 (HPA: antibody validation). Agreement with a positive control and expected compartment is therefore useful when interpreting an individual run.
Isoforms and epitope coverage	UniProt lists two SPRED2 isoforms and the WH1, KBD and SPR domains (UniProt Q7Z698). The supplied sources do not locate the catalog antibody's epitope or establish whether it detects both isoforms; avoid treating a difference between samples as an isoform difference without separate evidence.
IF/ICC Q&A: should nuclear fluorescence define the IHC result?	No. HPA calls nucleoplasm uncertain and cytosol supported in ICC-IF (HPA: subcellular ICC-IF); its tissue IHC summary is cytoplasmic (HPA: tissue IHC). Assess IF/ICC on its separate guide page rather than transferring an ICC-IF nuclear call into a paraffin-section IHC score.

Why is my staining missing, weak or wrong?
Situation	Likely cause	Next action
Expected positive cells stain weakly or remain blank.	A staining-run failure is possible; the supplied sources do not identify a SPRED2-specific retrieval condition or fixation sensitivity.	Confirm an identifiable HPA high-staining population, then review retrieval, antibody incubation and detection performance using routine run controls (HPA: tissue IHC; standard IHC practice).
Predominantly nuclear stain appears in IHC.	The observed compartment conflicts with the general cytoplasmic tissue pattern (HPA: tissue IHC); nuclear ICC-IF localisation is uncertain (HPA: subcellular ICC-IF).	Recheck morphology and counterstain, compare a positive control, and assess antibody-omission staining before scoring the nuclear signal (standard IHC practice).
Diffuse chromogen obscures cell boundaries.	Nonspecific staining or excessive detection signal may obscure a cellular pattern (standard IHC practice).	Check the antibody-omission control; review blocking, washes and chromogen development, then reassess whether staining resolves to cytoplasm (standard IHC practice; HPA: tissue IHC).
Unexpected cells stain more strongly than the reported positive population.	Cell identification, nonspecific antibody binding or endogenous detection activity may explain the contrast (standard IHC practice); HPA reports variable cytoplasmic levels (HPA: tissue IHC).	Verify the cell population on the counterstained section and inspect detection controls before calling cross-reactivity (standard IHC practice).
A low-staining tissue shows faint cytoplasmic signal.	Low does not mean absent: HPA reports low staining in parathyroid glandular cells and smooth muscle cells, among others (HPA: tissue IHC).	Record the cell type and intensity, and compare them with a reported high-staining control; do not use these low-staining tissues as proven negatives (HPA: tissue IHC).
Two assays give different nuclear and cytoplasmic impressions.	HPA's ICC-IF summary includes uncertain nucleoplasm and supported cytosol, while its tissue IHC summary is generally cytoplasmic (HPA: subcellular ICC-IF; tissue IHC).	Interpret each assay against its own evidence and controls; use the separate IF/ICC guide for fluorescence-specific decisions (HPA: subcellular ICC-IF; standard IHC practice).

Source: UniProt Q7Z698 Human Protein Atlas tissue Human Protein Atlas subcellular

Experiment evidence

IHC & IF images showing SPRED2 staining

IHC analysis of SPRED2 using anti-SPRED2 antibody (A06368-1). SPRED2 was detected in a paraffin-embedded section of human liver tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2 μg/ml rabbit anti-SPRED2 Antibody (A06368-1) overnight at 4°C. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using HRP Conjugated Rabbit IgG Super Vision Assay Kit (Catalog # SV0002) with DAB as the chromogen.

A06368-1 · IHCIHC analysis of SPRED2 using anti-SPRED2 antibody (A06368-1). SPRED2 was detected in a paraffin-embedded section of human liver tissue. Heat

IF analysis of SPRED2 using anti-SPRED2 antibody (A06368-1) and anti-Tubulin Alpha antibody (M03989-3). SPRED2 was detected in immunocytochemical section of U20S cell. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent (AR0022) for 15 mins. The cells were blocked with 10% goat serum. And then incubated with 5 μg/mL rabbit anti-SPRED2 Antibody (A06368-1) and mouse anti-Tubulin Alpha antibody (M03989-3) overnight at 4°C. Cy3 Conjugated Goat Anti-Rabbit IgG (BA1032) and DyLight®488 Conjugated Goat Anti-Mouse IgG (BA1126) were used as secondary antibody at 1:500 dilution and incubated for 30 minutes at 37°C. Visualize using a fluorescence microscope and filter sets appropriate for the label used.

A06368-1 · IFIF analysis of SPRED2 using anti-SPRED2 antibody (A06368-1) and anti-Tubulin Alpha antibody (M03989-3). SPRED2 was detected in immunocytoche

HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000198369-SPRED2/tissue/cerebral+cortex#imid_17855259

HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000198369-SPRED2/tissue/colon#imid_17855185

HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000198369-SPRED2/tissue/liver#imid_17855170

HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000198369-SPRED2/tissue/kidney#imid_17855171

HPA · ICC-IF CACO-2Human Protein Atlas immunofluorescence, CACO-2, antibody HPA038256; green = SPRED2, blue = nucleus, red = microtubules.

← swipe / scroll for more images →

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

Sample controls for SPRED2 IHC & IF

🧪Run kidney first: tubular cells should stain (HPA: High in cells in tubules). HPA detects SPRED2 in all 45 scored tissues and lists no negative tissue, so use no-primary and isotype controls; on the positive slide, compare tubular staining with background in other cell types without assuming those cells are SPRED2-negative (HPA: no negative tissue listed).

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

Negative control tissue: None in HPA: SPRED2 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 SPRED2 in CACO-2, U-251MG, U2OS, with annotated localisation: Nucleoplasm (uncertain) (HPA subcellular).

Technical controls: Include a no-primary, secondary-only slide and a host- and clonality-matched isotype control; use SPRED2-knockout material as a biological negative when available. Quench endogenous peroxidase for HRP detection, and account for endogenous biotin in kidney tubules if using biotin-based detection (datasheet: HRP/DAB detection in the selected IHC caption).

⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected A06368-1 paraffin-section caption does not state a fixative (selected-SKU tissue-IHC caption). That caption reports heat retrieval in EDTA at pH 8.0, but does not establish whether retrieval is required for every specimen (selected-SKU tissue-IHC caption). Paraffin IHC has a documented procedure here; the supplied evidence does not establish that frozen sections or IF are easier (selected-SKU tissue-IHC caption; HPA: ICC-IF images listed).

HPA tissue IHC evidence for SPRED2

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
Adrenal gland	Glandular cells	High	Protein (IHC)	HPA →
Duodenum	Glandular cells	High	Protein (IHC)	HPA →
Gallbladder	Glandular cells	High	Protein (IHC)	HPA →
Heart muscle	Cardiomyocytes	High	Protein (IHC)	HPA →
Kidney	Cells in tubules	High	Protein (IHC)	HPA →

Undetected expression · recommended negative controls

Tissue	Cell type	Level	Evidence	Source
None in HPA: SPRED2 is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead.

Section 3

Advanced SPRED2 IHC Tips

Troubleshoot SPRED2 staining in paraffin sections using the catalog antibody’s liver IHC conditions, then interpret results against tissue and subcellular evidence.

Which retrieval condition should I try first for weak SPRED2 staining?

Start with heat-mediated retrieval in EDTA at pH 8.0 for paraffin sections (datasheet A06368-1). The catalog antibody’s human liver image used that condition, followed by 10% goat serum blocking and 2 μg/ml primary antibody overnight at 4°C (caption A06368-1). If staining is weak, check section adhesion, retrieval temperature and heating time before changing the buffer; the caption does not specify a heating duration (caption A06368-1). Compare any adjusted condition with the original on adjacent sections, using a no-primary control to distinguish recovered signal from increased background (standard IHC practice).

Could fixation explain variable SPRED2 staining between paraffin blocks?

Target-specific SPRED2 fixation sensitivity is unknown; the selected liver caption identifies a paraffin section but does not report its fixative (caption A06368-1). Record each block’s fixative, fixation duration and processing history, then compare sections with the same pH 8.0 EDTA retrieval and 2 μg/ml primary concentration (caption A06368-1; standard IHC practice). If a block stains weakly, examine morphology and an independent control stain before changing retrieval or antibody exposure (standard IHC practice). Keep staining runs and detection times consistent when comparing blocks, because processing and assay variation can mimic a fixation effect (standard IHC practice).

Where should convincing SPRED2 staining appear in tissue sections?

Expect predominantly cytoplasmic staining in tissue IHC, while assessing any membrane-associated or vesicular pattern in the context of cell morphology (HPA tissue IHC: general cytoplasmic expression; UniProt Q7Z698: cell membrane and cytoplasmic vesicle membrane). SPRED2 has no transmembrane segment, so a crisp outline alone does not establish specific membrane localisation (UniProt Q7Z698 topology; standard IHC interpretation). The tissue IHC profile and cultured-cell imaging differ: the latter reports nucleoplasm as uncertain and cytosol as supported (HPA subcellular). Score cytoplasmic, membranous and nuclear signal separately, then check unusual patterns against a no-primary control and a second validated reagent where available (standard IHC practice).

How can epitope placement affect interpretation of SPRED2 staining?

SPRED2 has 2 reported isoforms, but the supplied antibody caption does not identify its epitope or establish isoform coverage (UniProt Q7Z698 isoforms; caption A06368-1). Obtain the catalog antibody’s immunogen sequence before interpreting discordant staining as an isoform difference (standard IHC practice). Map that sequence against the WH1 region (5–122), KBD (201–257) and SPR region (308–416), as well as both isoform sequences (UniProt Q7Z698 domains and isoforms). Phosphorylation is reported at residues 206, 228 and 231; whether those modifications affect this antibody requires direct epitope evidence and testing (UniProt Q7Z698 modified residues; standard IHC practice).

How should I investigate a conflicting SPRED2 immunofluorescence pattern?

Treat IF as a separate optimisation: the supplied catalog evidence documents chromogenic staining in a paraffin liver section, with no IF dilution or permeabilisation condition stated (caption A06368-1). Multiplex SPRED2 with a marker identifying the expected cell population, such as an epithelial marker when examining glandular cells reported as SPRED2 positive (HPA tissue IHC; standard IF practice). Choose spectrally separated fluorophores and assess an unstained section for tissue autofluorescence before assigning a weak SPRED2 signal (standard IF practice). Test permeabilisation if the epitope faces the cytosol or lies within vesicle-associated compartments; its orientation is unknown for this antibody (UniProt Q7Z698 localisation and topology; caption A06368-1).

What should I adjust when SPRED2 DAB staining is diffuse?

First compare a no-primary section with the stained section to identify secondary-reagent, endogenous peroxidase or chromogen background (standard IHC practice). The liver caption used 10% goat serum, 2 μg/ml primary overnight at 4°C, a peroxidase-conjugated secondary for 30 minutes at 37°C, and DAB (caption A06368-1). If background persists, verify the routine peroxidase block, shorten DAB development and assess wash adequacy while keeping a positive control beside the test section (standard IHC practice). Optimise primary concentration only after checking these steps, and preserve a record of exposure and development times for comparison (standard IHC practice).

How can I quantify SPRED2 IHC across samples? ⚠ ANSWER MARKED FOR VERIFICATION

Define the scored cell population and compartment before analysis; tissue IHC describes SPRED2 expression as generally cytoplasmic and variable (HPA tissue IHC). Use an H-score from staining intensity and the percentage of positive cells, or report positive-cell density per mm² when cellular distribution is the question (standard IHC practice). Normalise counts to eligible, intact cells or tissue area within matched regions, excluding necrosis, folds and section edges by a rule set before scoring (standard IHC practice). Keep retrieval, antibody concentration, DAB development, imaging settings and scoring thresholds consistent across samples; the catalog starting concentration is 2 μg/ml (caption A06368-1; standard IHC practice).

How do I distinguish genuine SPRED2 signal from staining artefacts?

Check whether staining follows intact cell boundaries and plausible cytoplasmic structures: tissue IHC reports general cytoplasmic expression, and UniProt places SPRED2 in cytoplasm and at membranes (HPA tissue IHC; UniProt Q7Z698 localisation). Strong glandular-cell staining is reported in several tissues, while the HPA tissue reliability assessment indicates only medium consistency with RNA expression (HPA tissue IHC: positive-cell list and Approved reliability). Treat staining confined to cut edges, necrotic regions or no-primary controls as suspect; assess endogenous peroxidase when DAB is diffuse (standard IHC practice). Interpret isolated nuclear staining cautiously because the cultured-cell nucleoplasmic assignment is uncertain (HPA subcellular).

Boster reagents

Best SPRED2 / Sprouty-related, EVH1 domain-containing protein 2 IHC Antibodies

A06368-1 has IHC data from human paraffin-embedded liver and IF data from U20S cells (catalog IHC/IF captions). Its listed reactivity covers human, mouse and rat (catalog reactivity).

Real IHC data

Anti-SPRED2 Antibody ®

Cat # A06368-1

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A06368-1 will render with human liver paraffin-section IHC using EDTA retrieval at pH 8.0 and DAB detection (A06368-1 IHC caption). The same SKU has an IF example in U20S cells and lists both IF and ICC applications (A06368-1 IF caption; catalog applications).

Which to pick: Choose A06368-1 for human tissue IHC: its own image documents a paraffin-embedded liver section, but the fixative is unreported (A06368-1 IHC caption). For IF/ICC, the same SKU has a U20S cell IF example and lists both applications (A06368-1 IF caption; catalog applications). For mouse or rat samples, A06368-1 lists reactivity with both species, while its supplied IHC image documents human tissue (catalog reactivity; A06368-1 IHC caption).

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

References

UniProt Consortium. UniProt entry Q7Z698 (SPRE2_HUMAN, Sprouty-related, EVH1 domain-containing protein 2).
Human Protein Atlas. SPRED2 tissue IHC expression (reliability: Approved).
Human Protein Atlas. SPRED2 subcellular location (ICC-IF): Mainly localized to the nucleoplasm. In addition localized to the cytosol..
Human Protein Atlas. SPRED2 antibody validation summary (2 antibodies).
Expression of Spred2 in the urothelial tumorigenesis of the urinary bladder. PloS one 2021 — PMC8612556.
SPRED2: A Novel Regulator of Epithelial-Mesenchymal Transition and Stemness in Hepatocellular Carcinoma Cells. International journal of molecular sciences 2023 — PMC10003366.
Neurofibromin Encoded by the Neurofibromatosis Type 1 (NF1) Gene Promotes the Membrane Translocation of SPRED2, Thereby Inhibiting the ERK Pathway in Breast Cancer Cells. International journal of molecular sciences 2025 — PMC12563662.
Evidence for downregulation of the negative regulator SPRED2 in clinical prostate cancer. British journal of cancer 2013 — PMC3593530.
PubMed PMID:15683364 — UniProt-cited evidence.
PubMed PMID:14702039 — UniProt-cited evidence.
PubMed PMID:15815621 — UniProt-cited evidence.