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CDC25C / M-phase inducer phosphatase 3 · IHC design guide
Design Immunohistochemistry for CDC25C
Plan chromogenic IHC for CDC25C around the general nuclear tissue pattern (HPA tissue IHC). The guide covers section handling, antibody selection, controls and scoring, with the low consistency between antibody staining and RNA expression in mind (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
CDC25C 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 | General nuclear tissue staining (HPA tissue IHC) | |
| Staining pattern | Nuclear staining across tissue cell types (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet M01343) | |
| Positive control | Adrenal gland+4 more · see all | |
| Negative control | Liver+3 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 | Staining and RNA show low consistency (HPA tissue IHC) | |
| Regulation | No expression regulator annotated (UniProt) | |
| Isoform / epitope | 5 isoforms; check epitope coverage (UniProt) |
Section 1
Recommended CDC25C IHC & IF Protocols
The catalog antibody uses EDTA pH 8.0 retrieval (datasheet M01343). These published CDC25C IHC protocols cover lung, esophageal, vulvar, and glioma specimens (PMC9952919; PMC3845600; PMC2887779; PMC12480179).
| Sample | Paraffin-embedded human stomach cancer tissue; fixative not specified (datasheet M01343) |
| Fixation | Image fixative and duration unreported (datasheet M01343); 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 M01343); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet M01343) |
| Primary antibody | Rabbit monoclonal (clone HIG-3) anti-CDC25C, 1:50 recommended; image 1:100 (datasheet M01343) |
| Primary incubation | Overnight at 4 °C (datasheet M01343) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet M01343) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | CDC25C-positive staining in glandular cells of adrenal gland (HPA tissue IHC: High). HPA tissue profile: General nuclear expression. No signal in the no-primary control. |
💡Decision noteStart with EDTA pH 8.0 heat retrieval for the catalog antibody (datasheet M01343). Citrate retrieval is documented for other CDC25C antibodies (PMC3845600; PMC2887779; PMC12480179).
Section 2
What Is the Expected CDC25C Staining Pattern?
CDC25C is annotated in the nucleus (UniProt P30307) and shows general nuclear staining in tissue IHC (HPA tissue IHC). Expect signal in cells such as adrenal glandular cells and cervical squamous epithelium, both scored high (HPA tissue IHC). HPA rates the tissue profile Approved but reports low consistency between antibody staining and RNA expression (HPA tissue IHC). CDC25C has no transmembrane segment (UniProt P30307 topology).
| Predominantly nuclear staining in adrenal glandular cells or cervical squamous epithelial cells. | This matches high staining in those cell types and HPA's general nuclear profile (HPA tissue IHC). Assess individual cells against their tissue architecture; a high score for one cell type does not mean every cell in that section must stain. |
| Strong staining mainly outside nuclei, with little nuclear signal. | This conflicts with the nuclear annotation (UniProt P30307) and general nuclear tissue pattern (HPA tissue IHC). Treat the compartment mismatch as a possible artefact; review morphology, detection controls and antibody validation before assigning it to CDC25C. |
| Strong signal in liver cholangiocytes, ovarian stromal cells, smooth muscle cells or soft tissue fibroblasts. | Those specific cell populations are scored not detected (HPA tissue IHC). Check cell identity and controls for cross-reactivity or endogenous detection activity. Their scores do not establish that every cell in the corresponding tissue is negative. |
| Broad, diffuse colour across cells or extracellular areas, obscuring nuclear boundaries. | The distribution does not support a nuclear call (UniProt P30307; HPA tissue IHC). In chromogenic IHC, excess detection background or insufficient blocking or washing can obscure localisation (general IHC practice). Judge a cell positive only when its staining is interpretable. |
| No visible staining in an expected high-staining cell population. | Adrenal glandular and cervical squamous epithelial cells are scored high (HPA tissue IHC). An absent result warrants a technical check, but HPA's Approved profile has low consistency with RNA expression (HPA tissue IHC); one unstained section alone does not establish target absence. |
💡Expected CDC25C appearanceCall a convincing positive result high, predominantly nuclear staining in an HPA high-scoring cell type, such as adrenal glandular cells (HPA tissue IHC); widespread cytoplasmic or extracellular colour without nuclear definition is suspect against the nuclear annotation (UniProt P30307).
| Tissue and cell selection | HPA scores adrenal glandular cells, caudate glia and cervical squamous epithelium high, but skeletal myocytes and splenic red pulp cells low (HPA tissue IHC). Select and score the named cell population, since a low-scoring population may give a faint reference signal. |
| Strength of the tissue evidence | The tissue profile is Approved, yet antibody staining and RNA expression have low consistency (HPA tissue IHC). Use its cell-level observations as expectations, not as proof that every sample or antibody must reproduce the same intensity. |
| Compartment and topology | CDC25C is nuclear and lacks a transmembrane segment (UniProt P30307). Nuclear enrichment supports interpretation; a membrane rim alone has no support from this topology. The supplied record does not define how much cytoplasmic signal is acceptable. |
| Isoforms and epitope coverage | UniProt lists 5 CDC25C isoforms and a rhodanese domain at residues 321–428 (UniProt P30307). The supplied evidence does not locate the catalog antibody's epitope or establish which isoforms it detects, so isoform-specific staining cannot be inferred. |
| Modification and processing | UniProt lists modified residues, including phosphoserines, and annotates no signal peptide or propeptide (UniProt P30307). The supplied evidence does not show that these features alter IHC staining or fixation sensitivity; avoid assigning a faint result to them. |
| IF/ICC Q&A: where should signal localise? | Mainly to nuclear speckles; HPA calls that location supported in ICC-IF (HPA subcellular). This finer pattern can guide IF image reading but does not require a speckled appearance in chromogenic tissue IHC, where HPA describes general nuclear expression (HPA tissue IHC). |
| Situation | Likely cause | Next action |
|---|---|---|
| Expected nuclei are blank in an HPA high-scoring cell type. | A staining workflow failure is possible; HPA also reports low staining–RNA consistency (HPA tissue IHC). | Confirm the named cells are present, then review the IHC-validated antibody, antigen retrieval, detection reagents and a working positive control (general IHC practice). Interpret absence cautiously rather than declaring CDC25C absent. |
| Nuclear staining is weak and hard to score. | The chosen population may have a low HPA score, as for skeletal myocytes or splenic red pulp cells (HPA tissue IHC). | Compare with a high-scoring population, such as adrenal glandular cells (HPA tissue IHC), under the same staining run. Score only signal distinguishable from the local background (general IHC practice). |
| Strong colour appears mainly at cell borders or in cytoplasm. | The pattern conflicts with nuclear localisation and the absence of a transmembrane segment (UniProt P30307). | Recheck cell boundaries and nuclear counterstain, then compare primary-antibody omission and detection controls (general IHC practice). Do not count border-only colour as the expected pattern. |
| Broad colour persists across tissue, including spaces between cells. | Nonspecific chromogen deposition or endogenous detection activity may be contributing (general IHC practice). | Inspect a primary-antibody omission control; review blocking, washing and endogenous-activity control appropriate to the detection system (general IHC practice). Reassess nuclear signal only after background is controlled. |
| A cell type scored not detected stains strongly. | Cell identification, cross-reactivity or endogenous detection activity needs checking; HPA scores liver cholangiocytes and soft tissue fibroblasts not detected (HPA tissue IHC). | Verify the stained cells morphologically and compare detection controls (general IHC practice). Keep the HPA score tied to its named cell type, rather than extending it to the whole tissue. |
| Two antibodies give different tissue patterns. | HPA reports low consistency between antibody staining and RNA expression; CAB003800 is IHC Approved, while the supplied IHC status for HPA066991 is unspecified (HPA tissue IHC; HPA antibodies). | Check which antibody has IHC validation and compare nuclear localisation, named cell populations and controls on matched sections (general IHC practice). Do not treat ICC support alone as IHC validation (HPA antibodies). |
Experiment evidence
IHC & IF images showing CDC25C staining
M01343 · IHCIHC analysis of CDC25C using anti-CDC25C antibody (M01343). CDC25C was detected in a paraffin-embedded section of human stomach cancer tissu
M01343 · IHCIHC analysis of CDC25C using anti-CDC25C antibody (M01343). CDC25C was detected in a paraffin-embedded section of human lymph node tissue. H
M01343-2 · IHCM01343-2 staining MPIP3 in human testis sections by Immunohistochemistry (IHC-P -paraformaldehyde-fixed, paraffin-embedded sections). Tissue
PB9756 · IFIF analysis of Cdc25C using anti-Cdc25C antibody (PB9756) and anti-Tubulin Alpha antibody (M03989-3). Cdc25C was detected in immunocytochemi
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000158402-CDC25C/tissue/cerebral+cortex#imid_1286372
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000158402-CDC25C/tissue/colon#imid_1286298
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000158402-CDC25C/tissue/liver#imid_1286299
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000158402-CDC25C/tissue/kidney#imid_1286292
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody CAB003800; green = CDC25C, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody CAB003800; green = CDC25C, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for CDC25C IHC & IF
🧪Run adrenal gland first and score its glandular cells, which show High staining (HPA: High in adrenal gland glandular cells). Use liver cholangiocytes as the negative tissue, where CDC25C is not detected (HPA: Not detected in liver cholangiocytes); on the adrenal slide, treat any unstained neighboring cells as a background comparison rather than an established negative cell type.
Positive control tissue: Adrenal gland (Glandular cells, HPA High)
Negative control tissue: Liver (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show CDC25C in A-431, U-251MG, U2OS, CACO-2, SiHa, with annotated localisation: Nuclear speckles (supported) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control and a concentration-matched rabbit IgG isotype control for the rabbit primary antibody (caption: rabbit anti-CDC25C M01343). Use CDC25C knockout material or, if the immunizing peptide is available, a peptide-block control to assess specificity; quench endogenous peroxidase before HRP/DAB detection and inspect the adrenal section for tissue pigment or other background (caption: HRP/DAB detection).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected paraffin-section caption does not state a fixative (caption: fixative not stated). Heat-mediated EDTA retrieval at pH 8.0 was used for the illustrated paraffin section, but retrieval dependence has not been established (caption: EDTA pH 8.0). Frozen sections have no demonstrated ease advantage here; IF can assess the reported nuclear-speckle pattern, but no comparative ease evidence is supplied (HPA: nuclear speckles, supported).
HPA tissue IHC evidence for CDC25C
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Low 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 → |
| Caudate | Glial cells | High | Protein (IHC) | HPA → |
| Cerebellum | Cells in molecular layer | High | Protein (IHC) | HPA → |
| Cerebral cortex | Neuronal cells | High | Protein (IHC) | HPA → |
| Cervix | Squamous epithelial cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Liver | Cholangiocytes | Not detected | Protein (IHC) | HPA → |
| Ovary | Ovarian stroma cells | Not detected | Protein (IHC) | HPA → |
| Smooth muscle | Smooth muscle cells | Not detected | Protein (IHC) | HPA → |
| Soft tissue | Fibroblasts | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced CDC25C IHC Tips
Troubleshoot CDC25C staining in paraffin sections by checking retrieval, nuclear localisation, controls and scoring before interpreting chromogenic signal.
Which retrieval conditions should I try first for weak CDC25C staining?
Start with heat-mediated retrieval in EDTA at pH 8.0 for paraffin sections (datasheet M01343). The selected antibody detected CDC25C in a paraffin-embedded human stomach cancer section after that retrieval, followed by 1:100 primary antibody overnight at 4°C (datasheet M01343). Keep section thickness, heating and cooling conditions consistent across the comparison, and include a positive control on each run (standard IHC practice). If staining remains weak, vary retrieval duration on serial sections while checking tissue integrity; consider another buffer only as a fallback and validate it against the EDTA condition (standard IHC practice).
Could fixation explain weak or uneven CDC25C staining?
The selected tissue-IHC caption describes a paraffin-embedded section but does not state its fixative, so CDC25C-specific fixation sensitivity is unknown (datasheet M01343). Record the fixative and fixation duration for each specimen, then compare sections processed and stained together with the same retrieval and detection conditions (standard IHC practice). Overfixation, underfixation and uneven fixation can change antigen accessibility or tissue morphology in IHC generally, but their effect on this antibody has not been established here (standard IHC practice; datasheet M01343). If signal varies within a specimen, examine morphology and processing history before attributing the pattern to CDC25C expression (standard IHC practice).
How should I assess nuclear and cytoplasmic CDC25C staining?
Prioritise nuclear staining when assessing CDC25C in chromogenic sections: UniProt assigns it to the nucleus, and HPA reports general nuclear tissue expression (UniProt P30307 subcellular location; HPA tissue IHC). HPA subcellular imaging places it mainly in nuclear speckles, although individual speckles may not be resolvable in routine chromogenic IHC (HPA subcellular; standard IHC practice). Compare DAB signal with a nuclear counterstain and score nuclei and cytoplasm separately when both appear stained (standard IHC practice). Diffuse cytoplasmic colour without convincing nuclear signal warrants checks of background, retrieval and the no-primary control before a biological interpretation (standard IHC practice).
Can isoforms or phosphorylation alter what this antibody detects in tissue?
CDC25C has 5 annotated isoforms, 19 listed modified residues and a rhodanese domain spanning residues 321–428 (UniProt P30307). The supplied caption does not identify the antibody epitope or establish which isoforms it recognises, so staining cannot be assigned to one isoform from this IHC result alone (datasheet M01343; UniProt P30307). Phosphorylation is relevant to CDC25C biology, but these payloads do not show that it changes this antibody’s staining (UniProt P30307 function and modified residues; datasheet M01343). For an isoform-specific question, obtain epitope information and validate recognition with appropriate reference material before interpreting section-to-section differences (standard IHC practice).
How can I extend the tissue findings to a CDC25C multiplex IF experiment?
Treat the chromogenic paraffin result as an IHC starting point, then optimise antibody concentration and imaging controls for IF separately (datasheet M01343; standard IF practice). CDC25C is nuclear and has no transmembrane segment, so use permeabilisation that permits antibody access to nuclear epitopes, adjusting its strength to preserve morphology (UniProt P30307 subcellular location and topology; standard IF practice). Multiplex with a validated marker for the cell type being assessed, using the marker and a nuclear stain to assign CDC25C signal to individual cells (standard IF practice). Choose fluorophores away from the specimen’s strongest autofluorescence and include single-colour and no-primary controls to assess bleed-through and background (standard IF practice).
What should I check when DAB staining is widespread or patchy?
First compare the stained slide with a no-primary control and inspect whether colour follows tissue edges, folds or damaged regions (standard IHC practice). The selected caption used a peroxidase-conjugated secondary antibody and DAB, so include an endogenous peroxidase block as a general chromogenic workflow step and verify that the block works in the specimen (datasheet M01343; standard IHC practice). Its 10% goat serum block and 1:100 primary dilution are documented starting conditions for this antibody (datasheet M01343). If background persists, titrate the primary antibody, review wash stringency and shorten chromogen development while retaining a positive control (standard IHC practice).
How should I score CDC25C in paraffin sections consistently? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and region of interest before scoring, and exclude folds, necrosis and poorly preserved areas (standard IHC practice). Because nuclear expression is expected, report the percentage of positive nuclei and, when intensity is reproducible, a nuclear H-score calculated as percentage at each intensity multiplied by its 0–3 score (UniProt P30307 subcellular location; standard IHC practice). Alternatively, count positive nuclei per mm² and normalise that count to evaluable tissue area or total nuclei in the same region (standard IHC practice). Keep retrieval, DAB development, thresholds and observer rules consistent, and report cytoplasmic staining separately if it is present (standard IHC practice).
How can I distinguish genuine CDC25C signal from staining artefacts?
A credible result should show staining in identifiable cells with a nuclear component, consistent with UniProt’s nuclear assignment and HPA’s general nuclear IHC profile (UniProt P30307 subcellular location; HPA tissue IHC). HPA lists high staining in adrenal gland glandular cells and no detection in liver cholangiocytes, but it also reports low consistency between antibody staining and RNA expression; use such patterns as context, not proof of specificity (HPA tissue IHC). Compare suspected positives with matched negative controls and inspect edges, necrotic regions and endogenous enzyme activity before counting them (standard IHC practice). Interpret differences cautiously when morphology, retrieval or detection conditions differ between sections (standard IHC practice).
Boster reagents
Best CDC25C / M-phase inducer phosphatase 3 IHC Antibodies
Anti-CDC25C antibodies include human paraffin-section IHC examples and a human-cell IF example (figure captions); the IF catalog antibody lists human, mouse and rat reactivity (catalog: PB9756).
Real IHC data
Anti-Cdc25C Rabbit Monoclonal Antibody
Cat # M01343
Real IHC data
Anti-MPIP3 Antibody
Cat # M01343-2
Real IF data
Anti-Cdc25C Antibody ®
Cat # PB9756
M01343 shows IHC in paraffin-embedded human stomach cancer tissue, lymph node and stomach (image captions); M01343-2 shows IHC-P in formaldehyde-fixed, paraffin-embedded human testis (image caption). PB9756 shows IF/ICC in human U2OS cells (image caption) and lists human, mouse and rat reactivity (catalog: PB9756).
Which to pick: For tissue IHC, choose M01343, a rabbit monoclonal with human paraffin-section examples using EDTA retrieval; its fixative is unreported (catalog and image captions: M01343). M01343-2 is a mouse monoclonal alternative with a paraffin-section human testis IHC-P example using citrate retrieval (catalog and image caption: M01343-2). For IF/ICC, choose PB9756 based on its U2OS IF example; it also lists mouse and rat reactivity, although the supplied IF example is human (catalog and image caption: PB9756). The selected M01343 tissue-IHC caption documents paraffin sections, but does not specify the fixative (selected-SKU IHC image M01343).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P30307 (MPIP3_HUMAN, M-phase inducer phosphatase 3).
- Human Protein Atlas. CDC25C tissue IHC expression (reliability: Approved).
- Human Protein Atlas. CDC25C subcellular location (ICC-IF): Mainly localized to the nuclear speckles..
- Human Protein Atlas. CDC25C antibody validation summary (2 antibodies).
- CDC25C Protein Expression Correlates with Tumor Differentiation and Clinical Outcomes in Lung Adenocarcinoma. Biomedicines 2023 — PMC9952919.
- Overexpression of Cdc25C predicts response to radiotherapy and survival in esophageal squamous cell carcinoma patients treated with radiotherapy followed by surgery. Chinese journal of cancer 2013 — PMC3845600.
- Overexpression of CDC25B, CDC25C and phospho-CDC25C (Ser216) in vulvar squamous cell carcinomas are associated with malignant features and aggressive cancer phenotypes. BMC cancer 2010 — PMC2887779.
- Cell division cycle 25 C (CDC25C) mediates cell-cycle progression and immune evasion in glioma. Discover oncology 2025 — PMC12480179.
- PubMed PMID:2195549 — UniProt-cited evidence.
- PubMed PMID:11078813 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for CDC25C (M-phase inducer phosphatase 3). Expected localisation General nuclear tissue staining (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet M01343). Validated IHC antibody: M01343 at 1:50 recommended; image 1:100 (the product's own IHC validation image is shown). Controls (HPA): positive Adrenal gland (Glandular cells); negative Liver. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human stomach cancer tissue; fixative not specified (datasheet M01343); 2. Fixation: Image fixative and duration unreported (datasheet M01343); 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 retrieval: EDTA pH 8.0 (datasheet M01343); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet M01343); 8. Primary antibody: Rabbit monoclonal (clone HIG-3) anti-CDC25C, 1:50 recommended; image 1:100 (datasheet M01343); 9. Primary incubation: Overnight at 4 °C (datasheet M01343). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific); Caveat: Staining and RNA show low consistency (HPA tissue IHC); Isoform / epitope: 5 isoforms; check epitope coverage (UniProt); Regulation: No expression regulator annotated (UniProt). Evidence: UniProt P30307, HPA tissue IHC · Approved, HPA ICC-IF · Mainly localized to the nuclear speckles., Datasheet M01343, PMC9952919, PMC3845600. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/CDC25C/immunohistochemistry-ihc-guide.