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CCL25 / C-C motif chemokine 25 · IHC design guide
Design Immunohistochemistry for CCL25
Plan chromogenic CCL25 IHC-P using the catalog antibody's 1:50–1:200 dilution range (datasheet). Use small-intestinal glandular cells as a positive reference for cytoplasmic staining (HPA tissue IHC), and account for secretion when interpreting the pattern (UniProt).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
CCL25 Immunohistochemistry Experimental Design Guide
Expected localisation, validated protocols, controls and antibodies — the at-a-glance facts below, then the full design guide.
Must know before staining
| Expected localisation | Cytoplasmic staining in a few tissues (HPA tissue IHC) | |
| Staining pattern | Cytoplasmic staining in small-intestinal glandular cells (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen) | |
| Positive control | Small intestine | |
| Negative control | Adipose tissue+4 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent across sections (standard IHC practice; not target-specific) | |
| Caveat | Secretion may separate protein staining from RNA location (HPA tissue IHC) | |
| Regulation | No expression regulator specified (UniProt) | |
| Isoform / epitope | 3 isoforms; signal peptide 1–23 is removed; no cytoplasmic domain (UniProt) |
Section 1
Recommended CCL25 IHC & IF Protocols
The catalog antibody’s IHC-P protocol is followed by published CCL25 IHC methods for mouse heart and human ileal sections (PMC5009347; PMC9536268).
| Sample | Paraffin-embedded Mouse large intestine tissue; fixative not specified (datasheet A04077) |
| Fixation | Image fixative and duration unreported (datasheet A04077); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat-induced epitope retrieval in citrate buffer, pH 6.0, 20 min at 95–98 °C (standard rule: cytoplasmic / membrane antigen) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% normal serum of the secondary host, 30 min, room temperature (standard) |
| Primary antibody | Rabbit anti-CCL25, 1:50-1:200 (datasheet A04077) |
| Primary incubation | Overnight at 4 °C (standard) |
| Detection | HRP-polymer secondary, DAB chromogen 5–10 min (standard) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | CCL25-positive staining in glandular cells of small intestine (HPA tissue IHC: Medium). HPA tissue profile: Cytoplasmic expression in a few tissues. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6.0 HIER at 95–98 °C for 20 min (page antigen retrieval); the human ileal study used EDTA pH 9 (PMC9536268).
Section 2
What Is the Expected CCL25 Staining Pattern?
CCL25 is a secreted protein with no transmembrane segment (UniProt O15444). In paraffin tissue sections, expect cytoplasmic staining in small-intestinal glandular cells, where HPA reports medium staining; duodenal glandular cells show low staining (HPA tissue IHC). HPA describes expression in a few tissues and rates its tissue IHC evidence Approved, while cautioning that the tissue locations of RNA and secreted protein may differ (HPA tissue IHC).
| Medium cytoplasmic staining in small-intestinal glandular cells. | This matches the reported positive IHC pattern (HPA tissue IHC: small intestine, glandular cells, Medium). Score cell type and compartment together; secretion can complicate assignment of protein to its producing cell (UniProt O15444; HPA tissue IHC reliability note). |
| Strong nuclear-only or sharply membrane-bound staining. | Neither is the reported cytoplasmic pattern (HPA tissue IHC). A membrane-bound interpretation is especially doubtful because CCL25 has no transmembrane segment (UniProt O15444). Check morphology and staining controls before calling either pattern specific. |
| Prominent staining in appendix glandular cells or bronchial respiratory epithelial cells. | Both are listed as Not detected by tissue IHC (HPA tissue IHC). Consider cross-reactivity or endogenous chromogenic activity, especially if control sections share the pattern (general IHC practice). |
| Uniform color across cells, stroma, and section edges. | This lacks the cell-specific glandular pattern reported in small intestine (HPA tissue IHC). Diffuse background can arise from nonspecific reagent binding, incomplete blocking, or detection chemistry (general IHC practice); do not score it as CCL25-positive cells. |
| No staining in an adequately preserved small-intestine section. | This conflicts with the reported medium glandular staining (HPA tissue IHC). First check the antibody's IHC validation and the detection run; a negative slide alone cannot establish absent CCL25, particularly for a secreted protein (HPA tissue IHC reliability note; general IHC practice). |
💡Expected CCL25 appearanceCall a section positive when small-intestinal glandular cells show discernible medium cytoplasmic staining (HPA tissue IHC); isolated strong nuclear or membrane-only color, or uniform color outside those cells, does not match that reported pattern (HPA tissue IHC; UniProt O15444 topology).
| Secreted location and tissue assignment | CCL25 is secreted and lacks a transmembrane segment (UniProt O15444). HPA warns that RNA and protein tissue locations may differ for secreted proteins (HPA tissue IHC); interpret extracellular color with morphology rather than assigning it automatically to the nearest cell. |
| Positive and lower-signal comparisons | Small-intestinal glandular cells are Medium, whereas duodenal glandular cells are Low by HPA tissue IHC. These are observed staining levels, not prescribed controls or a guarantee that every section will have the same intensity (HPA tissue IHC; general IHC practice). |
| Signal peptide and antibody epitope | The signal peptide spans residues 1–23 and the annotated CCL25 chain spans 24–150 (UniProt O15444). The supplied HPA validation summary does not identify the antibody epitope (HPA antibodies); infer neither precursor recognition nor mature-chain recognition from the staining pattern. |
| Isoforms and antibody specificity | UniProt lists 3 isoforms, with isoform 2 described as an antagonist of isoform 1 (UniProt O15444). The supplied HPA antibody summary reports IHC Approved but gives no isoform-specific validation (HPA antibodies); tissue color cannot distinguish isoforms. |
| IF/ICC: Is cellular localization established? | No HPA ICC-IF images or main cellular location are supplied for CCL25 (HPA subcellular). The secreted annotation supports biological context, but the tissue IHC pattern cannot validate an IF/ICC localization claim (UniProt O15444; HPA tissue IHC). |
| Situation | Likely cause | Next action |
|---|---|---|
| Small-intestine glandular cells are blank. | The result disagrees with HPA's Medium glandular staining; the failed step is unknown from appearance alone (HPA tissue IHC). | Review tissue identity and morphology, then check antibody labeling, retrieval conditions, detection reagents, and a run control (general IHC practice). Treat retrieval changes as general optimization, not a known CCL25 fixation effect. |
| Only duodenum is faint. | Low glandular staining is reported for duodenum (HPA tissue IHC). | Compare with small intestine, where glandular staining is reported as Medium (HPA tissue IHC). Record the weaker duodenal result before increasing detection intensity, which can also increase background (general IHC practice). |
| Appendix glandular cells show widespread color. | HPA reports those cells as Not detected; specific CCL25 staining is therefore uncertain (HPA tissue IHC). | Check a detection-only control for endogenous activity and assess whether color tracks tissue structures or section-wide background (general IHC practice). Reassess the result against small-intestine glandular staining (HPA tissue IHC). |
| Nuclei dominate the signal. | Nuclear staining does not match HPA's cytoplasmic tissue description (HPA tissue IHC). | Inspect the counterstain and detection-only control, then rescore only clearly localized chromogenic signal (general IHC practice). Do not assign a nuclear CCL25 location from this slide. |
| Color coats tissue edges or appears throughout stroma. | A broad deposit does not reproduce HPA's glandular cytoplasmic pattern; secretion also limits simple cell-of-origin claims (HPA tissue IHC; UniProt O15444). | Inspect washing, blocking, chromogen development, and section quality (general IHC practice). Keep extracellular color separate from scored glandular cytoplasmic staining. |
| A positive-looking slide has no clean cellular boundaries. | Overlapping background can obscure the glandular pattern, regardless of the antibody's Approved IHC status (HPA antibodies; general IHC practice). | Compare a detection-only control and an HPA-listed Not detected tissue, then adjust general staining or imaging conditions before scoring intensity (HPA tissue IHC; general IHC practice). |
Experiment evidence
IHC & IF images showing CCL25 staining
A04077 · IHCImmunohistochemistry analysis of CCL25 in paraffin-embedded Mouse large intestine tissue using CCL25 pAb at a dilution of 1:300 (40x lens).
A04077 · IHCImmunohistochemistry analysis of CCL25 in paraffin-embedded Rat large intestine tissue using CCL25 pAb at a dilution of 1:300 (40x lens).
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000131142-CCL25/tissue/cerebral+cortex#imid_15436587
HPA · Small intestineHuman Protein Atlas IHC, Small intestine. Source: https://www.proteinatlas.org/ENSG00000131142-CCL25/tissue/small+intestine#imid_15436128
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000131142-CCL25/tissue/colon#imid_15436161
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000131142-CCL25/tissue/liver#imid_15436154
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for CCL25 IHC & IF
🧪Run small intestine first; its glandular cells should stain (HPA: Medium in small-intestine glandular cells). Run adrenal gland as the negative comparator (HPA: Not detected in adrenal glandular cells); on the positive slide, assess non-glandular cells for background-level staining, while interpreting any extracellular signal cautiously because CCL25 is secreted (UniProt O15444: Secreted).
Positive control tissue: Small intestine (Glandular cells, HPA Medium)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA carries no ICC-IF cell line for CCL25; 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-species-, isotype-, and clonality-matched control immunoglobulin controls, plus CCL25-knockout tissue if available or an immunizing-peptide block when the peptide is available (standard IHC practice). In small intestine, control endogenous peroxidase for chromogenic detection and check tissue autofluorescence if using IF (standard IHC/IF practice).
⚠️Feasibility: A target-specific fixation window and fixation effect are unreported in the supplied evidence; the selected A04077 paraffin-section caption does not report a fixative (selected A04077 tissue-IHC caption). Antigen-retrieval dependency is also unreported, so optimize retrieval on paraffin sections rather than assume a required condition (selected A04077 tissue-IHC caption; standard IHC practice). The evidence does not establish frozen sections or IF/ICC as easier (HPA subcellular: no ICC-IF cell-line images); in small intestine, distinguish glandular staining from possible extracellular signal from a secreted chemokine and from endogenous peroxidase or tissue autofluorescence (UniProt O15444: Secreted; standard IHC/IF practice).
HPA tissue IHC evidence for CCL25
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Secreted protein, tissue location of RNA and protein is expected to differ.). 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 |
|---|---|---|---|---|
| Small intestine | Glandular cells | Medium | 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 → |
| Appendix | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Breast | Adipocytes | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced CCL25 IHC Tips
For CCL25 chromogenic IHC on paraffin sections, interpret staining in light of its secretion, limited tissue distribution, and unreported antibody epitope (UniProt O15444; HPA tissue IHC).
What retrieval should I use for CCL25 staining in paraffin sections?
Start with citrate buffer at pH 6.0 for heat-induced retrieval at 95–98 °C for 20 minutes (page retrieval rule). Allow sections to cool in buffer, then keep deparaffinisation, detection, and imaging conditions identical across the comparison (standard IHC practice). The catalog image shows staining in paraffin-embedded mouse large intestine at 1:300, but its caption does not report a retrieval method (A04077 caption). If staining is weak, test a different retrieval buffer on an adjacent section as a fallback and compare signal against background (standard IHC practice). Do not attribute an improvement to CCL25 epitope chemistry without an epitope map (record: no epitope supplied).
How should I investigate weak CCL25 staining when fixation varies between blocks?
CCL25-specific fixation sensitivity is unknown; the selected mouse large-intestine caption says paraffin-embedded but does not state a fixative (A04077 caption). Record each block’s fixative and fixation duration before comparing staining, because these processing differences can alter antigen accessibility in IHC (standard IHC practice). For a prospective pilot, compare matched samples fixed in 10% neutral buffered formalin for 6 and 24 hours, followed by identical processing and retrieval (standard IHC practice). Score signal and background in the same anatomical compartment, using adjacent sections where possible (standard IHC practice). Treat any difference as a result of that controlled pilot, not an established CCL25-specific fixation effect (record: no target-specific fixation evidence).
Should CCL25 appear inside glandular cells or outside them?
Expect possible cytoplasmic staining in a limited set of tissues: HPA reports medium staining in small-intestinal glandular cells and low staining in duodenal glandular cells (HPA tissue IHC). CCL25 has a signal peptide at residues 1–23, a mature chain at 24–150, and no transmembrane segment (UniProt O15444 topology). Secreted protein may also appear outside producing cells, so extracellular deposits alone cannot identify the source cell (UniProt O15444 secreted; HPA reliability description). Assess glandular-cell staining and adjacent extracellular signal separately, alongside section morphology and a no-primary control (standard IHC practice). Avoid calling a sharp plasma-membrane rim the expected CCL25 pattern without independent validation (UniProt O15444 topology).
Can this stain distinguish CCL25 isoforms or detect the signal peptide?
The record lists 3 CCL25 isoforms, but supplies no antibody epitope or isoform-specific validation (UniProt O15444; record: no epitope supplied). Isoform 2 antagonises isoform 1, so functional conclusions cannot follow from a total CCL25 stain (UniProt O15444 function). The annotated signal peptide spans residues 1–23 and the mature chain spans 24–150; staining cannot establish which sequence the antibody recognises (UniProt O15444 processing; record: no epitope supplied). Ask for an epitope map or compare independently validated isoform reagents before assigning an isoform to a positive cell (standard IHC validation practice). Report results as CCL25 immunoreactivity until that specificity has been established (record: no isoform-specific antibody evidence).
How can I use IF to check the cellular source of an IHC signal?
For a separate IF experiment, multiplex CCL25 with a glandular epithelial marker such as pan-cytokeratin in small intestine, where HPA reports glandular-cell staining (HPA tissue IHC; standard marker practice). Choose a far-red CCL25 fluorophore and inspect an unstained section, because tissue autofluorescence can confound dim signals (standard IF practice). The antibody epitope is unreported: use mild permeabilisation, such as 0.1% Triton X-100 for 5 minutes, only when testing an intracellular epitope; omit it when testing an accessible extracellular epitope (record: no epitope supplied; standard IF practice). Compare single-channel controls before judging overlap (standard IF practice). The paraffin-section IHC caption does not establish IF performance or an IF fixation method (A04077 caption).
What should I change when CCL25 IHC stains most of the section?
First compare the test section with a no-primary control and inspect whether colour follows tissue edges, folds, necrosis, or the expected glandular compartment (standard IHC practice; HPA tissue IHC). For peroxidase detection, include an endogenous-peroxidase block before DAB and check whether residual colour persists without primary antibody (standard chromogenic IHC practice). Titrate from the reported 1:300 antibody dilution against a weaker concentration while holding retrieval and exposure to DAB constant (A04077 caption; standard IHC practice). Wash thoroughly and review blocking and secondary-antibody controls if diffuse staining persists (standard IHC practice). Widespread staining deserves extra scrutiny because HPA reports cytoplasmic expression in only a few tissues (HPA tissue IHC).
How should I quantify CCL25 staining across matched tissue sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the anatomical region and cell class before scoring; HPA identifies small-intestinal glandular cells as a positive compartment (HPA tissue IHC). For cellular staining, record the percentage of positive glandular cells and an H-score using intensity categories 0–3 across matched fields (standard IHC scoring practice). For extracellular deposits, measure positive area per mm² of viable tissue separately from the cellular score (UniProt O15444 secreted; standard image-analysis practice). Normalise cell counts to all evaluable cells of the same class, and area measurements to the corresponding viable tissue area (standard IHC quantification practice). Keep retrieval, antibody dilution, DAB development, thresholds, and section selection consistent across groups (standard IHC practice).
How can I distinguish a credible CCL25 signal from staining artefacts?
A credible result should reproduce in the relevant compartment on adjacent sections and exceed the no-primary control under matched processing (standard IHC validation practice). HPA reports medium cytoplasmic staining in small-intestinal glandular cells, while CCL25 is secreted and lacks a transmembrane segment (HPA tissue IHC; UniProt O15444 topology). Examine staining restricted to cut edges or necrotic regions as a possible processing artefact, and check diffuse DAB colour against an endogenous-peroxidase control (standard IHC practice). Do not assign extracellular staining to the nearest cell without supporting cell-specific evidence, because secreted proteins can move from their source (UniProt O15444 secreted; HPA reliability description). Report unexpected cell types or membrane rims descriptively until independently validated (standard IHC practice).
Boster reagents
Best CCL25 / C-C motif chemokine 25 IHC Antibodies
A04077 has IHC images from paraffin-embedded mouse and rat large intestine (image captions) and lists human reactivity and IF as an application (catalog). No IF image is supplied (catalog).
Real IHC data
Anti-C-C motif chemokine 25 CCL25 Antibody
Cat # A04077
A04077 will render with an IHC figure from paraffin-embedded mouse large intestine at 1:300 (image caption). Its applications include IHC and IF, its listed reactivity is human, and a second IHC caption shows rat large intestine at 1:300 (catalog; image captions).
Which to pick: Choose A04077 for paraffin-section IHC because its own images show mouse and rat large intestine; the fixative is unreported (image captions). For IF/ICC, A04077 lists IF and human reactivity, but the payload supplies no IF image or ICC claim (catalog). For cross-species work, the mouse and rat IHC images support only those pictured samples; the listed reactivity is human, so verify performance in the intended species (image captions; catalog).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry O15444 (CCL25_HUMAN, C-C motif chemokine 25).
- Human Protein Atlas. CCL25 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. CCL25 subcellular location (ICC-IF): Secreted.
- Human Protein Atlas. CCL25 antibody validation summary (1 antibodies).
- Chemokine CCL25 Induces Migration and Extracellular Matrix Production of Anulus Fibrosus-Derived Cells. International journal of molecular sciences 2018 — PMC6121557.
- The chemokine receptor CCR9 is required for the T-cell-mediated regulation of chronic ileitis in mice. Gastroenterology 2011 — PMC3086928.
- Abrogation of CC chemokine receptor 9 ameliorates ventricular remodeling in mice after myocardial infarction. Scientific reports 2016 — PMC5009347.
- CCR9 axis inhibition enhances hepatic migration of plasmacytoid DCs and protects against liver injury. JCI insight 2022 — PMC9536268.
- PubMed PMID:9285413 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
- PubMed PMID:15057824 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for CCL25 (C-C motif chemokine 25). Expected localisation Cytoplasmic staining in a few tissues (HPA tissue IHC). Antigen retrieval: Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen). Validated IHC antibody: A04077 at 1:50-1:200 (the product's own IHC validation image is shown). Controls (HPA): positive Small intestine (Glandular cells); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded Mouse large intestine tissue; fixative not specified (datasheet A04077); 2. Fixation: Image fixative and duration unreported (datasheet A04077); verify before use.; 3. Sectioning: 4–5 µm sections on charged slides (standard); 4. Deparaffinisation: Xylene, graded ethanol series to water (standard); 5. Antigen retrieval: Heat-induced epitope retrieval in citrate buffer, pH 6.0 , 20 min at 95–98 °C…; 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% normal serum of the secondary host, 30 min , room temperature (standard); 8. Primary antibody: Rabbit anti-CCL25, 1:50-1:200 (datasheet A04077); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific); Caveat: Secretion may separate protein staining from RNA location (HPA tissue IHC); Isoform / epitope: 3 isoforms; signal peptide 1–23 is removed; no cytoplasmic domain (UniProt); Regulation: No expression regulator specified (UniProt). Evidence: UniProt O15444, HPA tissue IHC · Approved, HPA ICC-IF · Secreted, Datasheet A04077, PMC5009347, PMC9536268. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/CCL25/immunohistochemistry-ihc-guide.