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CDC25B / M-phase inducer phosphatase 2 · IHC design guide
Design Immunohistochemistry for CDC25B
Plan CDC25B chromogenic IHC in paraffin sections using the observed nuclear and cytoplasmic tissue pattern (HPA tissue IHC). This guide highlights germinal center cells as a high-staining reference (HPA tissue IHC) and the catalog antibody’s 2–5 μg/mL IHC range (datasheet PB9488).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
CDC25B 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 | Nuclear and cytoplasmic tissue staining (HPA tissue IHC) | |
| Staining pattern | Nuclear/cytoplasmic staining, most abundant in GI and lymphoid tissues (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet PB9488) | |
| Positive control | Lymph node+4 more · see all | |
| Negative control | Adrenal gland+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 | Antibody staining has medium consistency with RNA data (HPA tissue IHC) | |
| Regulation | Expression regulation is not annotated (UniProt) | |
| Isoform / epitope | 4 isoforms; check epitope coverage across variants (UniProt) |
Section 1
Recommended CDC25B IHC & IF Protocols
The catalog antibody’s IHC-P protocol (datasheet: PB9488) accompanies four published CDC25B chromogenic IHC protocols (PMC8435635; PMC2887779; PMC11555179; PMC8981514).
| Sample | Paraffin-embedded human stomach cancer tissue; fixative not specified (datasheet PB9488) |
| Fixation | Image fixative and duration unreported (datasheet PB9488); 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 PB9488); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet PB9488) |
| Primary antibody | Rabbit anti-CDC25B, 2-5μg/ml (datasheet PB9488) |
| Primary incubation | Overnight at 4 °C (datasheet PB9488) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet PB9488) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | CDC25B-positive staining in germinal center cells of lymph node (HPA tissue IHC: High). HPA tissue profile: Nuclear and cytoplasmic expression in several tissue types, most abundant in the gastrointestinal tract and lymphoid tissues. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA pH 8.0 retrieval (datasheet: PB9488); the vulvar carcinoma protocol also uses EDTA pH 8.0 for CDC25B (PMC2887779).
Section 2
What Is the Expected CDC25B Staining Pattern?
In paraffin-section IHC, expect nuclear and cytoplasmic CDC25B staining across several tissues, especially gastrointestinal and lymphoid tissues (HPA tissue IHC: Enhanced; medium consistency with RNA data). Germinal center cells are a strong reference population (HPA tissue IHC: High). Cytoplasmic staining is consistent with CDC25B localization at centrosomes and spindle poles (UniProt P30305: subcellular location). CDC25B has no transmembrane segment (UniProt P30305: topology).
| Strong nuclear and/or cytoplasmic staining in lymph-node germinal center cells. | This fits a useful positive reference (HPA tissue IHC: High in germinal center cells). Compare the cell population and compartment with the counterstain; the reference describes population-level staining, not a required signal in every cell. |
| Moderate staining in rectal glandular cells, renal glomerular cells, or splenic red-pulp cells. | These are plausible positive results in the named cell populations (HPA tissue IHC: Medium in each). Do not score the whole organ as uniformly positive: the reported levels apply to specific cells. |
| Staining restricted to cell surfaces or extracellular material, with no credible intracellular signal. | Treat this as a possible artefact: reported tissue staining is nuclear and cytoplasmic (HPA tissue IHC: profile), and CDC25B has no transmembrane segment (UniProt P30305: topology). Review morphology and controls before interpreting it as CDC25B. |
| Strong staining in cells reported as undetected, or widespread diffuse chromogen over several cell types. | For example, adrenal glandular cells and bronchial respiratory epithelial cells are reported as not detected (HPA tissue IHC: Not detected). Unexpected cellular staining may reflect cross-reactivity; diffuse deposit may reflect endogenous detection activity or background. Neither alone proves target expression. |
| No staining in a lymph-node germinal center cell population that is identifiable on the section. | This conflicts with the reported high signal (HPA tissue IHC: High in germinal center cells). Check that tissue and morphology are suitable, then review the IHC workflow and antibody controls before calling the specimen CDC25B negative. |
💡Expected CDC25B appearanceA convincing positive is high nuclear and/or cytoplasmic signal in lymph-node germinal center cells (HPA tissue IHC: High; nuclear and cytoplasmic profile); cell-surface-only or diffuse, noncellular signal warrants artefact review (UniProt P30305: no transmembrane segment).
| Tissue and cell selection | CDC25B has low RNA tissue specificity, yet IHC levels vary by cell population (HPA tissue IHC: low RNA tissue specificity; High in germinal center cells; Not detected in adrenal glandular cells). Compare like cells when interpreting a section. |
| Compartment and cell-cycle context | CDC25B acts in mitotic progression and is reported at centrosomes and spindle poles (UniProt P30305: function; subcellular location). HPA also reports nuclear and cytoplasmic tissue staining (HPA tissue IHC: profile); do not require visible spindle-pole staining in routine chromogenic IHC. |
| IHC evidence strength | The tissue profile is rated Enhanced, with medium consistency between staining and RNA expression (HPA tissue IHC: reliability). Two listed antibodies have Enhanced IHC validation (HPA antibodies: HPA038892 and HPA038893); this supports the reported pattern without validating every specimen or antibody. |
| Isoforms and epitope interpretation | Four isoforms are listed (UniProt P30305: isoforms). Their presence does not establish which isoforms an unspecified antibody detects; consult its documented immunogen or epitope before attributing a staining difference to an isoform. |
| Intracellular topology | CDC25B has no signal peptide, propeptide, or transmembrane segment (UniProt P30305: processing; topology). A membrane-rim or secreted-looking pattern therefore needs closer scrutiny; these annotations alone cannot identify the source of an unexpected stain. |
| IF/ICC comparison | For the separate IF/ICC guide, HPA describes mainly vesicular staining and additional mitotic-spindle staining, both uncertain (HPA subcellular: uncertain). The listed ICC validation is uncertain for HPA038893 and HPA075215 (HPA antibodies: ICC status); do not impose that IF pattern on tissue IHC. |
| Situation | Likely cause | Next action |
|---|---|---|
| Known-positive lymph node appears blank. | Possible assay failure, tissue-quality issue, or an unsuitable antibody condition; high germinal center staining is expected (HPA tissue IHC: High). | Confirm germinal centers on the counterstain, run a positive control, and check the antibody's documented IHC-P conditions. As general IHC practice, verify retrieval and detection steps before interpreting a true negative. |
| Signal is weak in a low-expression tissue. | Low staining can agree with the reference, such as thyroid glandular cells or lung alveolar cells (HPA tissue IHC: Low). | Compare with a high-reference cell population, such as lymph-node germinal center cells (HPA tissue IHC: High). Score intensity in the specified cells rather than treating weak signal as automatic assay failure. |
| Unexpected strong staining appears in an HPA-undetected cell population. | Cross-reactivity or endogenous detection activity is possible; for example, bronchial respiratory epithelial cells are not detected (HPA tissue IHC: Not detected). | Inspect a reagent-omission control and tissue morphology. If the result persists, compare an independent IHC-validated antibody with the same cell population; HPA lists two Enhanced IHC antibodies (HPA antibodies: HPA038892, HPA038893). |
| Brown deposit is broad, granular, or obscures cell boundaries. | Diffuse background may arise from nonspecific binding, endogenous detection activity, or excessive chromogen development (general IHC practice). | Review blocking, washes, detection controls, and development time (general IHC practice). Reassess whether signal is intracellular and cell-associated, as described for tissue staining (HPA tissue IHC: nuclear and cytoplasmic profile). |
| Only a cell-surface rim is visible. | The localization conflicts with intracellular tissue staining (HPA tissue IHC: profile) and the absence of a transmembrane segment (UniProt P30305: topology). | Check the counterstain, section edges, and negative controls; confirm an intracellular pattern with an independently validated IHC antibody before scoring the rim as CDC25B. |
| IF/ICC shows vesicles or a mitotic spindle: should tissue IHC match it? | HPA marks both IF/ICC locations uncertain (HPA subcellular: vesicles; mitotic spindle). Tissue IHC is reported as nuclear and cytoplasmic (HPA tissue IHC: profile). | Judge paraffin-section IHC against its tissue and cell-type references (HPA tissue IHC: profile and levels). Assess IF/ICC on its own guide page, with its uncertain localization evidence (HPA subcellular: summary). |
Experiment evidence
IHC & IF images showing CDC25B staining
PB9488 · IHCIHC analysis of Cdc25B using anti-Cdc25B antibody (PB9488). Cdc25B was detected in a paraffin-embedded section of human stomach cancer tissu
PB9488 · IFIF analysis of Cdc25B using anti-Cdc25B antibody (PB9488). Cdc25B was detected in an immunocytochemical section of U2OS cells. Enzyme antige
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000101224-CDC25B/tissue/cerebral+cortex#imid_13928576
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000101224-CDC25B/tissue/colon#imid_13928502
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000101224-CDC25B/tissue/liver#imid_13928503
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000101224-CDC25B/tissue/kidney#imid_13928488
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA038893; green = CDC25B, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA038893; green = CDC25B, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for CDC25B IHC & IF
🧪Run lymph node first and require staining in germinal center cells (HPA: High in lymph node germinal center cells). Use adrenal gland glandular cells as a negative tissue (HPA: Not detected); on the lymph node slide, cells outside germinal centers should show little specific signal relative to germinal center cells, but their antigen-negative status is unverified (HPA: High in germinal center cells only).
Positive control tissue: Lymph node (Germinal center cells, HPA High)
Negative control tissue: Adrenal gland (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show CDC25B in A-431, A-549, U2OS, PC-3, with annotated localisation: Vesicles (uncertain) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control and a concentration-matched, irrelevant rabbit IgG isotype control (datasheet caption: rabbit primary; standard IHC practice). Use CDC25B knockout material if available, or peptide competition if the immunizing peptide is available; quench endogenous peroxidase and check lymph node background before HRP/DAB detection (standard IHC practice; datasheet caption: HRP/DAB).
⚠️Feasibility: A target-specific fixation window and fixation effect are unreported, and the selected PB9488 paraffin-section caption does not state the fixative (datasheet caption: PB9488). That caption uses heat retrieval in EDTA at pH 8.0, so it supports starting with that retrieval condition but does not establish that retrieval is required; neither frozen sections nor IF are established as easier by the supplied evidence (datasheet caption: PB9488; HPA: ICC-IF images). In lymph node, assess endogenous peroxidase background when interpreting HRP/DAB staining (standard IHC practice; datasheet caption: HRP/DAB).
HPA tissue IHC evidence for CDC25B
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — Medium consistency between antibody staining and RNA expression data.). Rows are taken directly from the HPA tissue chart — click any row's HPA link to view the source.
Positive expression · recommended positive controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Lymph node | Germinal center cells | High | Protein (IHC) | HPA → |
| Breast | Myoepithelial cells | Medium | Protein (IHC) | HPA → |
| Cerebral cortex | Endothelial cells | Medium | Protein (IHC) | HPA → |
| Colon | Endothelial cells | Medium | Protein (IHC) | HPA → |
| Kidney | Cells in glomeruli | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adrenal gland | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | Not detected | Protein (IHC) | HPA → |
| Caudate | Glial cells | Not detected | Protein (IHC) | HPA → |
| Cerebellum | Cells in granular layer | Not detected | Protein (IHC) | HPA → |
| Cervix | Glandular cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced CDC25B IHC Tips
Use the paraffin-section protocol as the starting point, then assess staining against CDC25B localisation, cell-type controls, and the limits of the available evidence.
Which retrieval conditions should I start with for CDC25B paraffin sections?
Start with heat-mediated retrieval in EDTA at pH 8.0 (datasheet PB9488). The selected paraffin-section image used this condition before incubation with 2 μg/mL catalog antibody overnight at 4°C (datasheet PB9488). If staining is weak, check that sections were fully deparaffinised and that heating was consistent across the rack before changing retrieval conditions (standard IHC practice). Compare any adjusted retrieval run with the original EDTA condition on adjacent sections, because stronger DAB staining alone cannot establish CDC25B specificity (standard IHC practice). Score the same cell population in both runs; germinal center cells offer a documented high-expression reference (HPA: High in lymph-node germinal center cells).
Could fixation explain weak CDC25B staining in my paraffin sections?
Target-specific fixation sensitivity is unknown: the selected tissue image identifies a paraffin-embedded section but does not state its fixative (datasheet PB9488). Record the fixative, fixation duration, and processing history for each specimen before attributing a weak result to fixation (standard IHC practice). Compare specimens processed together and include a documented positive cell population, such as lymph-node germinal center cells, when material is available (HPA: High in germinal center cells). If signal differs across batches, repeat EDTA retrieval at pH 8.0 and the 2 μg/mL primary concentration consistently before testing fixation as an explanation (datasheet PB9488; standard IHC practice).
Should CDC25B staining appear in nuclei, cytoplasm, or mitotic structures?
Examine cytoplasmic staining and, in mitotic cells, staining near spindle poles or centrosomes; these compartments are annotated for CDC25B (UniProt P30305: subcellular location). Nuclear and cytoplasmic tissue staining has also been reported, with greatest abundance in gastrointestinal and lymphoid tissues (HPA: tissue IHC profile). Mark nuclei with a counterstain and inspect individual cells at high magnification before assigning a DAB deposit to a compartment (standard IHC practice). Treat a vesicular pattern cautiously because that location is marked uncertain in cell imaging, and use matched negative controls to assess diffuse staining (HPA: subcellular location; standard IHC practice).
Can this stain distinguish CDC25B isoforms or phosphorylation states?
Do not assign isoform identity from this chromogenic stain without antibody epitope information and isoform-specific validation (UniProt P30305: 4 isoforms; datasheet PB9488: tissue-IHC caption). CDC25B has a rhodanese domain at residues 431–538 and annotated phosphorylation sites, including serines 42 and 353 (UniProt P30305: domains and modified residues). Those annotations do not establish which sequence or modification the catalog antibody detects in paraffin sections (UniProt P30305; datasheet PB9488). If isoform or phosphorylation state matters, obtain the antibody's immunogen or epitope specification and validate discrimination with appropriate reference material before interpreting changes in DAB intensity (standard IHC practice).
How should I investigate CDC25B localisation with a complementary IF experiment?
Design IF as a separate assay: the selected antibody evidence here is from a paraffin-section chromogenic image (datasheet PB9488). In lymph node, multiplex CDC25B with a germinal center cell marker and a nuclear counterstain so cell identity and compartment can be judged together (HPA: High in germinal center cells; standard IF practice). Choose a far-red fluorophore when tissue autofluorescence obscures shorter-wavelength channels, and include single-label controls to assess channel bleed-through (standard IF practice). CDC25B has no transmembrane segment, so test permeabilisation for access to intracellular epitopes while checking that cellular structure is retained; the precise antibody epitope is unspecified here (UniProt P30305: topology and localisation; standard IF practice).
What should I check when CDC25B DAB staining is widespread?
Check a no-primary control to identify staining from the detection system, and inspect areas with endogenous pigment or damaged tissue separately (standard IHC practice). Block endogenous peroxidase before HRP and DAB detection; the selected image used a peroxidase-linked secondary and DAB, but does not specify a peroxidase-blocking step (datasheet PB9488; standard IHC practice). The image used 10% goat serum blocking and 2 μg/mL primary antibody overnight at 4°C (datasheet PB9488). If background persists, compare primary concentrations and wash conditions on adjacent sections while preserving a positive reference; widespread colour alone is not evidence of CDC25B expression (standard IHC practice).
How should I score heterogeneous CDC25B staining across tissue sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring: tissue IHC reports nuclear and cytoplasmic expression, while CDC25B also has cytoskeletal locations (HPA: tissue IHC profile; UniProt P30305: subcellular location). For a defined region, record the percentage of positive cells and staining intensity, then calculate an H-score only if intensity categories were set in advance (standard IHC practice). Normalise counts to the number of evaluable cells, or report positive-cell density per mm² of viable tissue, keeping nuclear and cytoplasmic results separate (standard IHC practice). Apply identical thresholds, counterstaining, and image settings across compared sections, and exclude folds or necrotic regions from the denominator (standard IHC practice).
How can I distinguish plausible CDC25B staining from artefact?
A plausible result follows identifiable cells and a defensible compartment: CDC25B is annotated at cytoplasmic cytoskeletal structures, and tissue IHC also reports nuclear and cytoplasmic staining (UniProt P30305: subcellular location; HPA: tissue IHC profile). Compare signal in germinal center cells, reported High, with adrenal glandular cells, reported Not detected, while accounting for tissue processing (HPA: tissue IHC levels; standard IHC practice). Discount staining confined to section edges, necrosis, or structures also coloured in a no-primary control; endogenous peroxidase can contribute to DAB signal (standard IHC practice). Interpret modest differences cautiously because tissue staining has only medium consistency with RNA expression data (HPA: Enhanced reliability description).
Boster reagents
Best CDC25B / M-phase inducer phosphatase 2 IHC Antibodies
PB9488 has IHC data from paraffin-embedded human stomach cancer tissue and IF/ICC data from U2OS cells (catalog image captions); listed reactivity covers human, mouse, and rat (catalog reactivity).
Real IHC data
Anti-Cdc25B Antibody ®
Cat # PB9488
PB9488 is listed for IHC and has an image from a paraffin-embedded human stomach cancer section (catalog applications; PB9488 IHC caption). PB9488 is also listed for IF/ICC and has an IF image from U2OS cells (catalog applications; PB9488 IF caption).
Which to pick: Choose PB9488 for paraffin-section IHC: its own caption documents heat retrieval in EDTA pH 8.0 and primary antibody at 2 μg/mL (PB9488 IHC caption). The same SKU supports IF/ICC selection through its U2OS image and listed IF/ICC applications (PB9488 IF caption; catalog applications). For other species, PB9488 lists mouse and rat reactivity, but the supplied IHC image shows human tissue; its clonality and the tissue fixative are unreported (catalog reactivity; catalog clone field; PB9488 IHC caption).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P30305 (MPIP2_HUMAN, M-phase inducer phosphatase 2).
- Human Protein Atlas. CDC25B tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. CDC25B subcellular location (ICC-IF): Mainly localized to vesicles. In addition localized to the mitotic spindle..
- Human Protein Atlas. CDC25B antibody validation summary (3 antibodies).
- Mechanism of Paeoniflorin on ANIT-Induced Cholestatic Liver Injury Using Integrated Metabolomics and Network Pharmacology. Frontiers in pharmacology 2021 — PMC8435635.
- 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.
- The BET inhibitor sensitivity is associated with the expression level of CDC25B in pancreatic cancer models. Cancer drug resistance (Alhambra, Calif.) 2024 — PMC11555179.
- Menadione reduces CDC25B expression and promotes tumor shrinkage in gastric cancer. Therapeutic advances in gastroenterology 2020 — PMC8981514.
- PubMed PMID:1836978 — UniProt-cited evidence.
- PubMed PMID:1662986 — UniProt-cited evidence.
- PubMed PMID:9188863 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for CDC25B (M-phase inducer phosphatase 2). Expected localisation Nuclear and cytoplasmic tissue staining (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet PB9488). Validated IHC antibody: PB9488 at 2-5μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Lymph node (Germinal center cells); negative Adrenal gland. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human stomach cancer tissue; fixative not specified (datasheet PB9488); 2. Fixation: Image fixative and duration unreported (datasheet PB9488); 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 PB9488); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet PB9488); 8. Primary antibody: Rabbit anti-CDC25B, 2-5μg/ml (datasheet PB9488); 9. Primary incubation: Overnight at 4 °C (datasheet PB9488). Watch for: Fixation: Keep fixation consistent across sections. (standard IHC practice; not target-specific); Caveat: Antibody staining has medium consistency with RNA data (HPA tissue IHC); Isoform / epitope: 4 isoforms; check epitope coverage across variants (UniProt); Regulation: Expression regulation is not annotated (UniProt). Evidence: UniProt P30305, HPA tissue IHC · Enhanced, HPA ICC-IF · Mainly localized to vesicles. In addition localized to the mitotic…, Datasheet PB9488, PMC8435635, PMC2887779. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/CDC25B/immunohistochemistry-ihc-guide.