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CCL25 / C-C motif chemokine 25 · IHC design guide
Design Immunohistochemistry for CCL25
Plan paraffin IHC for CCL25 using small-intestine glandular cells as a staining reference (HPA tissue IHC). This guide covers consistent fixation (standard IHC practice), antibody titration at 1:50–1:200 (datasheet), chromogenic controls (standard IHC practice), and interpretation of a secreted protein (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 | Medium cytoplasmic staining in small-intestine 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 | Secreted protein; RNA and protein locations may differ (HPA tissue IHC) | |
| Regulation | RNA enriched in intestine/lymphoid tissue (HPA tissue RNA) | |
| Isoform / epitope | 3 isoforms; mature chain starts at residue 24; epitope map unknown (UniProt) |
Section 1
Recommended CCL25 IHC & IF Protocols
Compare the catalog antibody’s IHC-P protocol (datasheet) with a published human ileal protocol (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 retrieval at 95–98 °C for 20 min (page retrieval); the 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 topology). In paraffin section IHC, the supplied HPA record shows cytoplasmic staining of medium intensity in small intestine glandular cells (HPA tissue IHC). HPA rates this tissue staining Approved and cautions that RNA and protein locations may differ for a secreted protein (HPA tissue IHC).
| Cytoplasmic stain in small intestine glandular cells. | This matches the observed positive pattern and medium intensity (HPA tissue IHC). Score the glandular cell compartment and compare its staining with nearby unstained structures. Because CCL25 is secreted, cytoplasmic staining identifies detectable protein in those cells; it does not establish that protein remains there or identify its extracellular destination (UniProt O15444; HPA tissue IHC). |
| Stain appears confined to nuclei or outlines cell membranes. | A dominant nuclear or membrane pattern does not match HPA's cytoplasmic tissue profile (HPA tissue IHC). UniProt describes CCL25 as secreted and lacking a transmembrane segment (UniProt O15444 topology). Check morphology, counterstain, detection controls and antibody conditions before interpreting this pattern as CCL25; localisation alone cannot establish the cause of a mismatch. |
| Strong stain appears in an HPA-listed negative cell population. | For example, adipocytes in adipose tissue and respiratory epithelial cells in bronchus are listed as not detected (HPA tissue IHC). Reproducible staining there warrants checks for cross-reactivity or endogenous detection activity. The HPA designation describes its observed assay result, so a negative listing is a comparison point rather than proof that every specimen must be unstained. |
| Colour is diffuse across cells, stroma or the section. | Widespread haze obscures the cell-associated cytoplasmic pattern reported by HPA (HPA tissue IHC). Secreted CCL25 may be present beyond producing cells, so extracellular colour alone cannot identify its source (UniProt O15444; HPA tissue IHC). Compare with a no-primary control and inspect whether background follows tissue edges, precipitate or the detection chemistry (general IHC practice). |
| Small intestine glandular cells show no convincing stain. | This conflicts with the medium glandular cell signal in the supplied HPA example (HPA tissue IHC), but one negative section does not establish absent CCL25. Check that the section contains intact glandular cells, then review the IHC antibody's validated conditions and run a suitable positive control (general IHC practice). HPA's Approved rating does not guarantee signal in every specimen (HPA tissue IHC). |
💡Expected CCL25 appearanceCall medium cytoplasmic staining in small intestine glandular cells the expected positive result (HPA tissue IHC); dominant nuclear, membrane-only or widespread nonspecific colour is outside that observed pattern (HPA tissue IHC; UniProt O15444 topology).
| Secretion and tissue distribution | CCL25 is secreted and has no transmembrane segment (UniProt O15444 topology). HPA explicitly warns that RNA and protein locations may differ for a secreted protein (HPA tissue IHC). Interpret staining at the protein level; RNA enrichment in intestine and lymphoid tissue does not specify which cells will stain in an IHC section (HPA tissue IHC). |
| Processing and isoforms | The precursor has a signal peptide at residues 1–23 and a CCL25 chain at residues 24–150; three isoforms are listed (UniProt O15444). Antibody epitope and isoform coverage are not supplied. These annotations therefore cannot predict whether this IHC antibody detects every isoform or whether processing changes its staining. |
| Tissue and antibody evidence | HPA reports medium staining in small intestine glandular cells and low staining in duodenum glandular cells (HPA tissue IHC). Its listed antibody, HPA055883, is IHC Approved; the supplied record does not label it Enhanced or provide a second-antibody confirmation (HPA antibody record). Use that evidence level when judging an unexpected pattern. |
| IF/ICC: can it confirm the compartment? | HPA summarises CCL25 as secreted but provides no main intracellular location or ICC-IF cell-line images, and the listed antibody has no ICC status in this payload (HPA subcellular; HPA antibody record). This record cannot establish an IF/ICC pattern or validate an IF protocol; the cytoplasmic expectation here comes from tissue IHC (HPA tissue IHC). |
| Situation | Likely cause | Next action |
|---|---|---|
| No signal in small intestine glandular cells. | The expected medium IHC signal is absent (HPA tissue IHC); the section, detection run or antibody conditions may need review (general IHC practice). | Confirm glandular morphology and run a positive control; review the IHC-validated antibody's supplied conditions and detection reagents (general IHC practice). |
| Apparent positive signal occurs only in nuclei or along membranes. | This differs from the reported cytoplasmic pattern (HPA tissue IHC). CCL25 has no transmembrane segment (UniProt O15444 topology); the image alone does not identify the artefact. | Recheck focal plane and counterstain, then compare a no-primary control and an HPA-pattern positive section (general IHC practice; HPA tissue IHC). |
| Listed negative tissue stains strongly. | Adipose tissue adipocytes and bronchus respiratory epithelial cells were not detected in HPA's examples (HPA tissue IHC); cross-reactivity or endogenous activity is possible. | Compare cell morphology with the HPA-listed population and inspect no-primary and detection controls; do not assign CCL25 solely from colour (general IHC practice). |
| Uniform brown haze masks cell boundaries. | Diffuse background prevents assessment of the reported cytoplasmic glandular pattern (HPA tissue IHC). Excess detection background or endogenous peroxidase activity are general IHC possibilities. | Inspect no-primary and detection-only controls; review blocking, washes and chromogen development using standard IHC practice. |
| Duodenum looks weaker than small intestine. | That direction agrees with HPA's low duodenum and medium small intestine glandular staining (HPA tissue IHC). Differences between sections still need matched processing for comparison (general IHC practice). | Score comparable glandular cells on sections processed together; record intensity by tissue without treating the lower duodenal signal as assay failure (HPA tissue IHC; general IHC practice). |
| An IF image seems inconsistent with the IHC pattern. | The supplied HPA subcellular record has no ICC-IF images or main location, and the listed antibody has no ICC status here (HPA subcellular; HPA antibody record). | Treat IF localisation as unverified by these payloads. Consult the separate IF/ICC guide for its controls; use the HPA tissue IHC pattern for this paraffin section interpretation (HPA tissue IHC). |
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 tissue (HPA: Not detected in adrenal glandular cells); on the positive slide, neighboring non-glandular cells should show only background signal, assessed against the no-primary control.
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 a no-primary, secondary-only control and a host-species-matched isotype control for a monoclonal primary or matched nonimmune IgG for a polyclonal primary (standard IHC practice). Use CCL25-knockout tissue or a peptide-block control if available; quench endogenous peroxidase for chromogenic IHC and check intestinal autofluorescence if performing IF (standard IHC/IF practice).
⚠️Feasibility: A target-specific fixation window, fixation effect, and antigen-retrieval dependency are unreported in the supplied evidence; the exact A04077 paraffin-section caption does not report a fixative (caption: mouse large intestine, 1:300). Relative ease of frozen sections versus IF/ICC is unreported, and HPA lists no ICC-IF cell-line images (HPA subcellular). In small intestine, distinguish glandular-cell staining from luminal background and possible extracellular signal (HPA: Medium in glandular cells; UniProt O15444: Secreted).
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
Use compartment, tissue context, and matched controls to troubleshoot chromogenic CCL25 staining in paraffin sections.
How should I retrieve CCL25 in paraffin sections when staining is weak?
Start with citrate buffer at pH 6.0 for heat induced retrieval at 95–98 °C for 20 minutes (page retrieval setting). After cooling, compare a stained section with a matched no primary control and confirm that any gain remains localized to plausible cells (general IHC practice). The catalog image shows CCL25 staining in paraffin embedded mouse large intestine at 1:300, but reports neither fixation nor retrieval conditions (A04077 caption). If signal remains weak, test a second retrieval condition on adjacent sections while holding antibody concentration and detection constant; excessive heating can damage morphology and make diffuse staining harder to interpret (general IHC practice).
Could fixation explain variable CCL25 staining between paraffin blocks?
CCL25 specific sensitivity to fixation is unknown from the supplied evidence; the catalog caption identifies paraffin embedded mouse large intestine but does not state its fixative (A04077 caption). Record each block's fixative, time before fixation, fixation duration, and processing history, then compare sections using the same retrieval and detection conditions (general IHC practice). For a controlled comparison, process adjacent specimens consistently and include a known positive section in each staining run (general IHC practice). Because CCL25 is secreted, evaluate both cell associated signal and nearby extracellular staining cautiously, without treating either pattern as proof of a fixation effect (UniProt O15444 subcellular location; general IHC practice).
Where should convincing CCL25 staining appear in chromogenic IHC?
Expect a predominantly cytoplasmic cellular pattern where tissue staining is detectable, while allowing for extracellular signal because CCL25 is secreted and has no transmembrane segment (HPA tissue IHC; UniProt O15444 topology). HPA reports medium staining in small intestinal glandular cells and low staining in duodenal glandular cells, useful contexts for assessing localization (HPA tissue IHC). Its tissue profile also notes that RNA and protein locations may differ for a secreted protein, so nearby deposited signal needs spatial interpretation (HPA tissue IHC). Compare cellular contours, adjacent extracellular areas, and a no primary control before calling broad stromal color specific (general IHC practice).
Can this antibody distinguish CCL25 isoforms or processed protein in tissue?
The record lists 3 CCL25 isoforms and a precursor with a signal peptide at residues 1–23; the annotated mature chain spans residues 24–150 (UniProt O15444 processing and isoforms). The supplied catalog image gives no epitope mapping, so staining alone cannot assign an isoform or distinguish precursor from secreted mature protein (A04077 caption; general IHC interpretation). This matters because isoform 2 is annotated as an antagonist of isoform 1 (UniProt O15444 function). Check the antibody's epitope documentation before making isoform claims, and use an independently isoform specific assay if that distinction is essential (general IHC practice).
How can I investigate CCL25 localization with multiplex IF?
Use IF as a separate localization experiment and validate the antibody under IF conditions, since the supplied catalog evidence is a paraffin section IHC image and HPA lists no ICC/IF cell images (A04077 caption; HPA subcellular). Pair CCL25 with a glandular epithelial marker when examining small intestine, where HPA reports medium glandular cell staining, and include single stain controls for each channel (HPA tissue IHC; general IF practice). Choose a fluorophore channel after measuring tissue autofluorescence on an unstained section (general IF practice). CCL25 has no transmembrane segment: use mild permeabilization to examine intracellular CCL25, or omit it when testing accessible extracellular deposits (UniProt O15444 topology; general IF practice).
How do I reduce diffuse brown signal without losing CCL25 staining?
First inspect a no primary control and an unstained section to separate antibody dependent color from pigment, endogenous enzyme activity, and tissue color (general IHC practice). Apply a peroxidase block before chromogenic detection, then optimize protein blocking, wash steps, and primary antibody concentration on adjacent sections (general IHC practice). The catalog paraffin image used 1:300 in mouse large intestine; treat that as an image condition rather than an established optimum for every block (A04077 caption). Because CCL25 is secreted, weak extracellular color may be biologically plausible, but diffuse edge concentrated staining or staining reproduced without primary antibody requires caution (UniProt O15444 subcellular location; general IHC practice).
What is a defensible way to quantify heterogeneous CCL25 IHC? ⚠ ANSWER MARKED FOR VERIFICATION
Define the tissue region and cell population before scoring, then report the percentage of positive glandular cells and an intensity based H-score when cellular staining is interpretable (general IHC practice; HPA tissue IHC). If extracellular staining is the endpoint, measure positive area or signal density per mm² within a predefined compartment instead of folding it into a cellular H-score (general IHC practice; UniProt O15444 subcellular location). Normalize cell counts to eligible cells and area measurements to analyzed tissue area, excluding folds and necrosis by a prespecified rule (general IHC practice). Keep imaging, threshold, and chromogen development settings consistent across sections (general IHC practice).
How can I distinguish true CCL25 signal from section artefacts?
A credible result should fit CCL25's secreted biology and the observed cytoplasmic staining profile, with small intestinal glandular cells providing a documented positive context (UniProt O15444 subcellular location; HPA tissue IHC). Treat strong isolated nuclear staining as unexpected because no nuclear location is supplied for CCL25 (UniProt O15444 subcellular location; HPA subcellular). Check whether color concentrates at cut edges, folds, or necrotic regions, and whether it persists in the no primary control or after an appropriate peroxidase block (general IHC practice). The catalog mouse large intestine image supports tissue staining at 1:300, but does not establish which individual cells or extracellular deposits are specific (A04077 caption).
Boster reagents
Best CCL25 / C-C motif chemokine 25 IHC Antibodies
A04077 lists human reactivity for IHC and IF (catalog: applications/reactivity). Its IHC images show paraffin-embedded mouse and rat large intestine (A04077 IHC image captions); no IF image is supplied (catalog: IF image alts).
Real IHC data
Anti-C-C motif chemokine 25 CCL25 Antibody
Cat # A04077
A04077 is listed for human IHC and IF (catalog: applications/reactivity). Its own IHC images show paraffin-embedded mouse and rat large intestine (A04077 IHC image captions); the catalog supplies no IF image (catalog: IF image alts).
Which to pick: For tissue IHC, choose A04077 for paraffin sections (A04077 IHC image captions); the fixative is unreported (A04077 IHC image captions). For IF, A04077 is a rabbit polyclonal antibody with IF listed, but no IF image or ICC application is supplied (catalog: host/applications/IF image alts; A04077 IHC image captions: pAb). For cross-species work, A04077 is cataloged as human-reactive, while mouse and rat appear in its IHC captions; confirm reactivity before using those species (catalog: reactivity; A04077 IHC image captions).
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: Secreted protein; RNA and protein locations may differ (HPA tissue IHC); Isoform / epitope: 3 isoforms; mature chain starts at residue 24 ; epitope map unknown (UniProt); Regulation: RNA enriched in intestine/lymphoid tissue (HPA tissue RNA). Evidence: UniProt O15444, HPA tissue IHC · Approved, HPA ICC-IF · Secreted, Datasheet A04077, PMC9536268. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/CCL25/immunohistochemistry-ihc-guide.