CDC25B / M-phase inducer phosphatase 2 · IHC design guide

Design Immunohistochemistry for CDC25B

Plan chromogenic IHC on paraffin sections around nuclear and cytoplasmic staining, with lymph node germinal center cells as a high-staining reference (HPA tissue IHC). The catalog antibody has an IHC-P starting range of 2–5 μg/mL (datasheet PB9488).

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

Immunohistochemistry protocol sheet for CDC25B (IHC for CDC25B): expected localisation Nuclear and cytoplasmic tissue staining (HPA tissue IHC), antibody PB9488, validated IHC image, and IHC protocol steps — Printable **CDC25B** IHC protocol sheet — expected localisation Nuclear and cytoplasmic tissue staining (HPA tissue IHC), antibody PB9488, controls and protocol steps. Open the full CDC25B IHC guide →

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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; high in germinal center cells (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	Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image PB9488)
	Caveat	Antibody staining and RNA show medium consistency (HPA tissue IHC)
	Regulation	Expression regulation unreported (UniProt)
	Isoform / epitope	4 isoforms; antibody epitope coverage is unspecified (UniProt; datasheet PB9488)

Section 1

Recommended CDC25B IHC & IF Protocols

Compare the catalog antibody’s EDTA pH 8.0 IHC protocol (datasheet: PB9488) with four published CDC25B chromogenic IHC methods (PMC8435635; PMC2887779; PMC11555179; PMC8981514).

Recommended immunohistochemistry (IHC-P) protocol parameters
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 method also used microwaved EDTA pH 8.0 (PMC2887779).

Section 2

What Is the Expected CDC25B Staining Pattern?

In paraffin-section IHC, expect CDC25B staining in nuclei and cytoplasm across several tissues, with high staining in lymph-node germinal center cells and medium staining in rectal glandular cells (HPA tissue IHC: Enhanced). Cytoplasmic staining is consistent with reported centrosome and spindle-pole localization (UniProt P30305). CDC25B has no transmembrane segment, so a dominant cell-surface pattern needs scrutiny (UniProt P30305 topology).

What am I looking at on my slide?
Germinal center cells stain strongly, with nuclear and cytoplasmic signal; rectal glandular cells stain less intensely.	This matches high germinal center and medium rectal glandular staining (HPA tissue IHC). Compare compartments within the identified cells; the HPA profile describes both nuclear and cytoplasmic expression (HPA tissue IHC).
A crisp cell-surface rim dominates while nuclei and cytoplasm show little signal.	A surface-dominant result is discordant with the nuclear and cytoplasmic IHC profile (HPA tissue IHC) and lack of a transmembrane segment (UniProt P30305 topology). Review morphology and antibody controls before calling it CDC25B.
Strong signal appears in bronchial respiratory epithelial cells or adrenal glandular cells.	Those specific cell populations were not detected in the HPA tissue survey (HPA tissue IHC). Consider cross-reactivity or endogenous chromogen-generating activity (general IHC practice); staining elsewhere in either organ requires separate assessment.
Brown color spreads across stroma, lumina, or most cells without clear cellular boundaries.	This is less interpretable than the cell-resolved nuclear and cytoplasmic profile (HPA tissue IHC). Background from detection reagents, endogenous activity, or incomplete blocking is possible (general IHC practice); check reagent controls.
Germinal center cells remain unstained in a section expected to be positive.	High staining is reported for these cells (HPA tissue IHC), so an absent result warrants an assay check. It does not alone establish that the specimen lacks CDC25B; verify cell identity and review assay controls (general IHC practice).

💡Expected CDC25B appearanceCall a result positive when identified germinal center cells show strong, cell-resolved nuclear and cytoplasmic staining (HPA tissue IHC: High); diffuse background or a dominant surface rim is discordant with that profile (HPA tissue IHC; UniProt P30305 topology).

How each factor affects the staining
Tissue and cell selection	Use identified lymph-node germinal center cells as the high-staining comparator; breast myoepithelial, rectal glandular, and several other listed cell populations are medium (HPA tissue IHC). An organ name alone is not a cell-level score.
Compartment and cell-cycle context	HPA describes nuclear and cytoplasmic tissue staining (HPA tissue IHC). UniProt places CDC25B at centrosomes and spindle poles and links it to G2/M progression (UniProt P30305); these annotations do not require a visible spindle in every paraffin section.
Strength of tissue evidence	The tissue-IHC reliability is Enhanced, with medium consistency between antibody staining and RNA data (HPA tissue IHC). HPA lists two antibodies with Enhanced IHC validation; interpret a new antibody's pattern against tissue and reagent controls, not that label alone.
Isoforms and modified residues	Four isoforms and several phosphorylated residues are annotated (UniProt P30305). Their effect on staining cannot be predicted without the antibody's epitope and validation details; avoid assigning a compartment or intensity difference to one isoform from this record.
Processing and topology	UniProt lists one chain spanning residues 1–580, with no signal peptide, propeptide, or transmembrane segment (UniProt P30305). There is no supplied basis to interpret surface staining as expected processing or shedding.

Why is my staining missing, weak or wrong?
Situation	Likely cause	Next action
No stain in the high-staining comparator	Germinal center cells are reported as High (HPA tissue IHC); absent staining could reflect specimen selection or an assay problem (general IHC practice).	Confirm germinal center morphology, then check the IHC-validated antibody's stated IHC-P procedure, reagent controls, retrieval, and detection steps (general IHC practice).
Signal is dominated by a membrane-like rim	That pattern conflicts with the reported nuclear and cytoplasmic IHC profile (HPA tissue IHC) and no-transmembrane topology (UniProt P30305).	Inspect the same cell types at higher magnification and compare reagent controls; withhold a CDC25B-positive call until the cellular pattern is supported (general IHC practice).
Unexpected cells stain strongly	Bronchial respiratory epithelial and adrenal glandular cells were not detected in the HPA survey (HPA tissue IHC); cross-reactivity or endogenous detection activity is possible (general IHC practice).	Check the precise cell identity, use appropriate detection controls, and compare with a known-positive section (general IHC practice).
Diffuse chromogen obscures compartments	Cell boundaries cannot be assessed against the nuclear and cytoplasmic reference profile (HPA tissue IHC); background may arise during blocking, washing, or detection (general IHC practice).	Review reagent-only controls, blocking and wash steps, and development time before scoring individual cells (general IHC practice).
Only one compartment appears positive	HPA reports both nuclear and cytoplasmic tissue expression, but its summary does not require both compartments in every cell (HPA tissue IHC).	Record nuclear and cytoplasmic staining separately, compare identified positive cells, and avoid treating one compartment alone as proof of artefact (HPA tissue IHC; general IHC practice).
Can the tissue-IHC pattern be used as an IF/ICC protocol?	HPA's ICC-IF summary places CDC25B mainly in vesicles and additionally on the mitotic spindle, with both locations marked uncertain (HPA subcellular ICC-IF).	Treat that localization as a tentative IF/ICC comparison, not an IHC-P staining requirement or protocol; use the separate IF/ICC guide for assay setup (HPA subcellular ICC-IF; HPA tissue IHC).

Source: UniProt P30305 Human Protein Atlas tissue Human Protein Atlas subcellular

Experiment evidence

IHC & IF images showing CDC25B staining

IHC analysis of Cdc25B using anti-Cdc25B antibody (PB9488). Cdc25B was detected in a paraffin-embedded section of human stomach cancer tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2 μg/ml rabbit anti-Cdc25B Antibody (PB9488) overnight at 4°C. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using HRP Conjugated Rabbit IgG Super Vision Assay Kit (Catalog # SV0002) with DAB as the chromogen.

PB9488 · IHCIHC analysis of Cdc25B using anti-Cdc25B antibody (PB9488). Cdc25B was detected in a paraffin-embedded section of human stomach cancer tissu

IF analysis of Cdc25B using anti-Cdc25B antibody (PB9488). Cdc25B was detected in an immunocytochemical section of U2OS cells. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent (AR0022) for 15 mins. The cells were blocked with 10% goat serum. And then incubated with 5 μg/mL rabbit anti-Cdc25B Antibody (PB9488) overnight at 4°C. DyLight®488 Conjugated Goat Anti-Rabbit IgG (BA1127) was used as secondary antibody at 1:500 dilution and incubated for 30 minutes at 37°C. The section was counterstained with DAPI. Visualize using a fluorescence microscope and filter sets appropriate for the label used.

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.

← 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 look for staining in germinal center cells (HPA: High in germinal center cells). Use adrenal gland glandular cells as a negative comparator (HPA: Not detected in adrenal gland glandular cells); on the lymph node slide, cells outside germinal centers should be assessed for background signal, without assuming they are CDC25B-negative (HPA: lymph node positive row identifies 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 no-primary (secondary-only) and concentration-matched rabbit IgG isotype controls matched to the primary antibody’s clonality, plus CDC25B-knockout material as a biological negative if available (standard IHC practice; caption: rabbit primary antibody). Quench endogenous peroxidase before HRP/DAB detection in lymph node sections (standard chromogenic IHC practice; caption: HRP/DAB detection).

⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected PB9488 tissue-IHC caption does not report a fixative (selected-SKU caption). The caption reports heat retrieval in EDTA at pH 8.0 for a paraffin section, but does not establish that retrieval is required across specimens; whether frozen sections or IF are easier is unreported (selected-SKU caption). Endogenous peroxidase can produce background with HRP/DAB detection in lymph node tissue, so interpret weak staining against the no-primary control (standard chromogenic IHC practice; caption: HRP/DAB detection).

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 catalog antibody’s paraffin-section example as a starting point, and interpret CDC25B staining alongside cell identity and tissue architecture (datasheet PB9488; HPA tissue IHC).

How should I adjust retrieval when CDC25B staining is weak in paraffin sections?

Start with heat-mediated retrieval in EDTA at pH 8.0 for paraffin sections (datasheet PB9488). Keep heating and cooling conditions consistent across slides, then compare a known positive compartment and a no-primary control before increasing retrieval intensity (standard IHC practice; HPA tissue IHC). The catalog example used 2 μg/ml primary antibody overnight at 4°C, so check antibody exposure and detection alongside retrieval (datasheet PB9488). If staining remains weak, test another retrieval condition on matched sections as a fallback, assessing tissue integrity and background rather than treating a stronger signal alone as proof of specificity (standard IHC practice).

Could fixation explain weak or uneven CDC25B staining?

CDC25B-specific sensitivity to fixation is unknown because the supplied paraffin-section caption does not state a fixative (datasheet PB9488). Record the fixative, fixation duration, processing history, and section age for each specimen, then compare matched sections under the same retrieval and staining conditions (standard IHC practice). If signal varies between blocks, assess preserved morphology and include a concurrently stained positive control before attributing the difference to CDC25B abundance (standard IHC practice). Do not infer fixation tolerance from CDC25B phosphorylation, its lack of a transmembrane segment, or HPA tissue staining; those records do not establish a fixation response (UniProt P30305; HPA tissue IHC).

Which cellular compartments should contain credible CDC25B staining?

Assess nuclear and cytoplasmic staining separately because both occur across tissues in HPA IHC (HPA tissue IHC). Cytoplasmic, centrosomal, and spindle-pole associations are also described for CDC25B, while the HPA subcellular record reports uncertain vesicular and mitotic-spindle locations (UniProt P30305; HPA subcellular). In chromogenic sections, inspect mitotic cells at high magnification and avoid calling a small brown focus a centrosome without independent spatial validation (standard IHC practice; UniProt P30305). Score staining within morphologically identified cells, using matched no-primary controls to distinguish compartmental signal from pigment or chromogen deposits (standard IHC practice).

Can this antibody distinguish CDC25B isoforms or phosphorylation states in tissue?

Treat the chromogenic result as total antibody-reactive CDC25B unless the recognized epitope and isoform reactivity have been established independently (standard IHC practice). UniProt lists 4 CDC25B isoforms and multiple phosphoserine sites, including residues 42, 169, 249, 323, and 353 (UniProt P30305). The supplied tissue caption gives staining conditions but no epitope map or phospho-specific validation, so intensity cannot identify an isoform or phosphorylation event (datasheet PB9488). For an isoform or modification claim, pair compartment-resolved IHC with an independently validated epitope-specific assay and matched controls (standard IHC practice).

How can IF help resolve ambiguous CDC25B localisation seen by chromogenic IHC?

Use IF as a separate validation experiment and multiplex CDC25B with a validated marker for the cell population being assessed, such as germinal center cells in lymph node (HPA tissue IHC; standard IF practice). Choose a spectrally separated, preferably far-red fluorophore after checking specimen autofluorescence, and include single-label and no-primary controls for channel bleed-through and background (standard IF practice). CDC25B has no transmembrane segment and has reported intracellular locations, so permeabilisation may be needed to access a cytoplasmic or nuclear epitope; the antibody’s exact epitope is unspecified (UniProt P30305; standard IF practice). Optimise fixation and permeabilisation for IF independently of the paraffin-section example, whose fixative was unreported (datasheet PB9488; standard IF practice).

How do I reduce diffuse brown staining without losing true CDC25B signal?

Compare a no-primary section with the stained section to identify secondary-reagent, endogenous-enzyme, and chromogen background (standard IHC practice). The catalog example used 10% goat serum blocking, a peroxidase-linked secondary, and DAB, but these conditions alone do not establish target specificity (datasheet PB9488). Include a peroxide block where appropriate for peroxidase detection, control DAB development time, and titrate primary concentration around the example’s 2 μg/ml while keeping detection exposure matched (standard IHC practice; datasheet PB9488). Judge improvement by preserved cell-associated staining and reduced signal in no-primary controls, particularly at section edges or damaged areas (standard IHC practice).

What is a defensible way to quantify CDC25B IHC across specimens? ⚠ ANSWER MARKED FOR VERIFICATION

Define the cell population and compartment before scoring because CDC25B IHC can be nuclear or cytoplasmic and tissue expression varies (HPA tissue IHC). For viable, morphologically comparable regions, report the percentage of positive cells and an H-score from intensity categories, with nuclear and cytoplasmic scores kept separate (standard IHC practice). Normalise cell counts to all evaluable cells of the same population, or report positive-cell density per mm² of viable tissue when area is the sampling unit (standard IHC practice). Fix the positivity threshold using controls and apply identical imaging, counterstain, and scoring rules across specimens (standard IHC practice).

How can I distinguish a CDC25B-positive cell from a staining artefact?

Prioritise reproducible nuclear or cytoplasmic cell-associated staining in intact tissue, consistent with the HPA tissue profile, while checking cell identity and morphology (HPA tissue IHC; standard IHC practice). Germinal center cells in lymph node provide a reported high-expression reference; adrenal glandular cells are reported as not detected and can inform a comparator when available (HPA tissue IHC). Distrust isolated signal in necrotic regions, section edges, or no-primary controls, and investigate diffuse brown deposits or residual endogenous-peroxidase activity before assigning positivity (standard IHC practice). A presumed vesicular or spindle pattern needs caution because those HPA subcellular assignments are uncertain, and chromogenic resolution may limit precise compartment calls (HPA subcellular; standard IHC practice).

Boster reagents

Best CDC25B / M-phase inducer phosphatase 2 IHC Antibodies

PB9488 has IHC data from paraffin-embedded human stomach cancer tissue (PB9488 IHC caption) and IF/ICC data from U2OS cells (PB9488 IF caption). Catalog reactivity lists human, mouse, and rat (PB9488 catalog).

Real IHC data

Anti-Cdc25B Antibody ®

Cat # PB9488

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PB9488 will render with an IHC figure from a paraffin-embedded human stomach cancer section (PB9488 IHC caption). Its IF/ICC figure shows U2OS cells, while the catalog lists human, mouse, and rat reactivity (PB9488 IF caption; PB9488 catalog).

Which to pick: Choose PB9488 for paraffin-section IHC: its own caption documents staining at 2 μg/ml, heat retrieval in EDTA at pH 8.0, and DAB detection; the fixative is unreported (PB9488 IHC caption). Choose the same SKU for IF/ICC because its own caption documents staining in U2OS cells at 5 μg/ml (PB9488 IF caption). For other species, PB9488 lists mouse and rat reactivity, but the supplied IHC and IF figures show human samples only; the catalog identifies a rabbit host and does not specify clonality (PB9488 catalog; PB9488 IHC and IF captions).

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.