CDC45 / Cell division control protein 45 homolog · IHC design guide

Design Immunohistochemistry for CDC45

Plan CDC45 staining in paraffin sections around the nuclear pattern seen in most tissues (HPA tissue IHC). Use testicular pachytene spermatocytes as a strong positive reference and compare nuclear staining across cell types (HPA tissue IHC).

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

Immunohistochemistry protocol sheet for CDC45 (IHC for CDC45): expected localisation Nuclear staining in most tissues (HPA tissue IHC), antibody M01367-2, validated IHC image, and IHC protocol steps — Printable **CDC45** IHC protocol sheet — expected localisation Nuclear staining in most tissues (HPA tissue IHC), antibody M01367-2, controls and protocol steps. Open the full CDC45 IHC guide →

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CDC45 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 staining in most tissues (HPA tissue IHC)
	Staining pattern	Nuclear; strongest in testicular pachytene spermatocytes (HPA tissue IHC)
	Antigen retrieval	EDTA pH 8.0 HIER, heat-mediated (datasheet M01367-2)
	Positive control ⓘ	Testis+3 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 paraffin sections. (standard IHC practice; not target-specific)
	Caveat	Staining and RNA show only medium consistency (HPA tissue IHC)
	Regulation	Highest in adult testis and thymus (UniProt)
	Isoform / epitope	3 isoforms; epitope coverage needs checking (UniProt)

Section 1

Recommended CDC45 IHC & IF Protocols

The catalog antibody protocol uses heat-mediated EDTA retrieval at pH 8.0 (datasheet: M01367-2). The published IHC methods below provide three tissue-based examples (PMC4714687; PMC7919231; PMC10956518).

Recommended immunohistochemistry (IHC-P) protocol parameters
Sample	Paraffin-embedded human ovarian cancer tissue; fixative not specified (datasheet M01367-2)
Fixation	Image fixative and duration unreported (datasheet M01367-2); 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 M01367-2); 20 min, 95–100 °C (standard)
Peroxidase block	3% H2O2, 10 min, room temperature (standard)
Blocking	10% goat serum (datasheet M01367-2)
Primary antibody	Mouse monoclonal (clone 6H6) anti-CDC45, 2μg/ml (datasheet M01367-2)
Primary incubation	Overnight at 4 °C (datasheet M01367-2)
Detection	Streptavidin-biotin complex (SABC), DAB chromogen (datasheet M01367-2)
Counterstain	Hematoxylin, blue, dehydrate and mount (standard)
Expected result	CDC45-positive staining in pachytene spermatocytes of testis (HPA tissue IHC: High). HPA tissue profile: Nuclear expression in most tissues, most abundant in testicular seminiferous duct. No signal in the no-primary control.

💡Decision noteStart with heat-mediated EDTA at pH 8.0 for the catalog antibody (datasheet: M01367-2); citrate at pH 6.0 is a published alternative to evaluate (PMC7919231).

Section 2

What Is the Expected CDC45 Staining Pattern?

CDC45 is a chromatin-associated nuclear protein with no transmembrane segment (UniProt O75419: subcellular location and topology). In paraffin-section IHC, expect nuclear staining, especially in testicular pachytene spermatocytes; lymphoid cells may stain at lower levels (HPA tissue IHC: High in pachytene spermatocytes; Medium in sampled lymphoid populations). HPA rates the tissue profile Enhanced, while reporting medium consistency between antibody staining and RNA expression (HPA tissue IHC: reliability).

What am I looking at on my slide?
Distinct nuclear stain in pachytene spermatocytes, with weaker stain in sampled lymphoid populations (HPA tissue IHC).	This matches the strongest listed IHC signal in testis and Medium staining in appendix, lymph node, and tonsil populations (HPA tissue IHC).
Predominantly cytoplasmic or membranous chromogen with little nuclear staining.	Review as a possible IHC artefact because CDC45 associates with chromatin and the HPA tissue profile is nuclear (UniProt O75419; HPA tissue IHC).
Strong stain in colon glandular cells or adrenal glandular cells.	Those sampled cells were Not detected by HPA; assess cross-reactivity or endogenous detection activity before calling them positive (HPA tissue IHC; standard IHC practice).
Hazy chromogen covers nuclei, cytoplasm, and surrounding tissue.	Diffuse background prevents a nuclear call; check blocking, antibody concentration, and wash conditions using standard IHC practice. HPA's CDC45 profile describes nuclear expression (HPA tissue IHC).
No discernible nuclear signal in testicular pachytene spermatocytes.	Treat the run as inconclusive until controls and the IHC workflow are checked; these cells are High in the HPA tissue profile (HPA tissue IHC; standard IHC practice).

💡Expected CDC45 appearanceCall CDC45 positive when chromogen marks nuclei of testicular pachytene spermatocytes strongly, with possible Medium nuclear staining in sampled lymphoid populations; diffuse or predominantly extranuclear stain alone is suspect (HPA tissue IHC; UniProt O75419: nuclear, chromatin-associated).

How each factor affects the staining
Cell compartment	Score the nuclear compartment in IHC: UniProt places CDC45 in the nucleus and on chromatin, and HPA reports nuclear expression across most tissues (UniProt O75419; HPA tissue IHC).
Choice of comparison cells	Testicular pachytene spermatocytes provide the strongest listed positive example; sampled colon and adrenal glandular cells are Not detected (HPA tissue IHC). Compare the specified cells, not an entire section.
Antibody evidence	Two listed antibodies, HPA000614 and CAB009459, have Enhanced IHC status; the tissue profile still has medium staining–RNA consistency (HPA antibodies; HPA tissue IHC). Interpret unexpected cells cautiously.
Isoform coverage	UniProt lists three CDC45 isoforms, but the payload gives no epitope or isoform recognition data for either antibody (UniProt O75419; HPA antibodies). Do not assign a staining difference to an isoform.
IF/ICC Q&A: where should signal appear?	Mainly in the nucleoplasm, with additional cytosol and centrosome localization reported in ICC-IF (HPA subcellular). This IF observation does not establish a cytoplasmic IHC pattern.

Why is my staining missing, weak or wrong?
Situation	Likely cause	Next action
The testis positive control has no nuclear stain.	A failed staining run is possible; pachytene spermatocytes are High in the reference profile (HPA tissue IHC).	Check control integrity, retrieval execution, primary-antibody application, and detection reagents using standard IHC practice; do not score samples from this run.
Chromogen appears mainly outside nuclei.	The pattern conflicts with nuclear, chromatin-associated CDC45 and HPA's nuclear tissue profile (UniProt O75419; HPA tissue IHC).	Inspect morphology and nuclear counterstain, then check blocking, washes, and detection controls using standard IHC practice.
Colon or adrenal glandular cells stain strongly.	These sampled cell types are Not detected in HPA tissue IHC; cross-reactivity or endogenous detection activity is possible (HPA tissue IHC; standard IHC practice).	Run a no-primary control and compare the stain with cell boundaries and the expected nuclear compartment (standard IHC practice; UniProt O75419).
Most of the section has diffuse color.	Background may obscure cell-specific nuclear stain; HPA describes nuclear expression (HPA tissue IHC; standard IHC practice).	Review blocking, wash stringency, and primary-antibody concentration; repeat alongside an appropriate control using standard IHC practice.
A low-staining tissue has no visible positive cells.	HPA reports Low staining in several sampled populations, including lung alveolar cells and stomach glandular cells (HPA tissue IHC).	Check a testis positive control and score only the relevant cell type; absence of a readily visible signal in a Low category alone does not establish reagent failure (HPA tissue IHC).
Two antibodies give different cell-level patterns.	Both listed antibodies have Enhanced IHC status, but the HPA tissue profile has medium staining–RNA consistency (HPA antibodies; HPA tissue IHC).	Compare matched sections and controls, then report the discrepancy by cell type and compartment rather than assigning an unsupported isoform explanation (UniProt O75419: three isoforms; standard IHC practice).

Source: UniProt O75419 Human Protein Atlas tissue Human Protein Atlas subcellular

Experiment evidence

IHC & IF images showing CDC45 staining

IHC analysis of CDC45L using anti-CDC45L antibody (M01367-2). CDC45L was detected in paraffin-embedded section of human ovarian cancer tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2μg/ml mouse anti-CDC45L Antibody (M01367-2) overnight at 4°C. Biotinylated goat anti-mouse IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) (Catalog # SA1021) with DAB as the chromogen.

M01367-2 · IHCIHC analysis of CDC45L using anti-CDC45L antibody (M01367-2). CDC45L was detected in paraffin-embedded section of human ovarian cancer tissu

IHC analysis of CDC45L using anti-CDC45L antibody (M01367-2). CDC45L was detected in paraffin-embedded section of human breast cancer tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2μg/ml mouse anti-CDC45L Antibody (M01367-2) overnight at 4°C. Biotinylated goat anti-mouse IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) (Catalog # SA1021) with DAB as the chromogen.

M01367-2 · IHCIHC analysis of CDC45L using anti-CDC45L antibody (M01367-2). CDC45L was detected in paraffin-embedded section of human breast cancer tissue

HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000093009-CDC45/tissue/cerebral+cortex#imid_223202

HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000093009-CDC45/tissue/colon#imid_223120

HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000093009-CDC45/tissue/liver#imid_223121

HPA · PancreasHuman Protein Atlas IHC, Pancreas. Source: https://www.proteinatlas.org/ENSG00000093009-CDC45/tissue/pancreas#imid_2834770

HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA000614; green = CDC45, blue = nucleus, red = microtubules.

← swipe / scroll for more images →

Real IHC / IF data behind the staining-pattern expectations in this section.

Sample controls for CDC45 IHC & IF

🧪Run testis first and look for staining in pachytene spermatocytes (HPA: High in pachytene spermatocytes); use adrenal glandular cells as the negative tissue (HPA: Not detected in adrenal glandular cells). On the testis slide, compare pachytene spermatocytes with surrounding cells that lack specific staining, without assuming every other cell type is CDC45-negative (HPA: High in pachytene spermatocytes).

Positive control tissue: Testis (Pachytene spermatocytes, HPA High)

Negative control tissue: Adrenal gland (HPA Not detected)

ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show CDC45 in A-431, U-251MG, U2OS, with annotated localisation: Nucleoplasm (supported) (HPA subcellular).

Technical controls: Include no-primary (secondary-only) and matched mouse isotype controls, plus CDC45-knockout tissue or cells as a biological negative where available (selected-SKU caption: mouse primary antibody; standard IHC controls). Block endogenous peroxidase and assess endogenous biotin background in testis because the reported detection uses a biotinylated secondary, streptavidin–biotin complex and DAB (selected-SKU caption: SABC/DAB detection).

⚠️Feasibility: No target-specific fixation window or fixation effect is reported, and the selected-SKU paraffin-section caption does not state a fixative (selected-SKU caption: fixative unreported). The reported IHC workflow uses heat retrieval in EDTA at pH 8.0, but the evidence does not establish whether retrieval is required or whether frozen sections are easier (selected-SKU caption: EDTA heat retrieval). ICC-IF images support evaluating nuclear localisation in a separate IF workflow, while testis background from the reported biotin-based detection needs control (HPA subcellular: nucleoplasm supported; selected-SKU caption: SABC/DAB detection).

HPA tissue IHC evidence for CDC45

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
Testis	Pachytene spermatocytes	High	Protein (IHC)	HPA →
Appendix	Lymphoid tissue	Medium	Protein (IHC)	HPA →
Lymph node	Germinal center cells	Medium	Protein (IHC)	HPA →
Tonsil	Non-germinal center cells	Medium	Protein (IHC)	HPA →

Undetected expression · recommended negative controls

Tissue	Cell type	Level	Evidence	Source
Adrenal gland	Glandular cells	Not detected	Protein (IHC)	HPA →
Caudate	Glial cells	Not detected	Protein (IHC)	HPA →
Cerebellum	Cells in granular layer	Not detected	Protein (IHC)	HPA →
Cerebral cortex	Glial cells	Not detected	Protein (IHC)	HPA →
Cervix	Glandular cells	Not detected	Protein (IHC)	HPA →

Section 3

Advanced CDC45 IHC Tips

Troubleshoot CDC45 staining in paraffin-section chromogenic IHC by checking retrieval, nuclear localisation, controls and scoring against the reported tissue pattern.

How should I retrieve CDC45 antigen when nuclear staining is weak?

Start with heat-mediated retrieval in EDTA at pH 8.0 for CDC45 paraffin-section IHC (datasheet M01367-2). The selected image used this retrieval before 2 µg/mL primary antibody overnight at 4 °C (datasheet M01367-2). Keep heating and cooling conditions consistent across adjacent sections when comparing retrieval outcomes (standard IHC practice). If nuclear staining remains weak, test another retrieval condition as a fallback while holding antibody concentration and detection constant (standard IHC practice). Judge any gain against tissue damage and background, using the expected predominantly nuclear pattern as a guide (HPA tissue IHC: nuclear expression in most tissues).

Can fixation explain weak CDC45 staining in paraffin sections?

The selected CDC45 paraffin-section caption does not report a fixative, so target-specific fixation sensitivity is unknown (datasheet M01367-2: fixative not stated). Record each specimen’s fixation history and compare sections processed under matched conditions before assigning weak staining to fixation (standard IHC practice). Include a known staining control, such as testis with pachytene spermatocytes, in the same run (HPA tissue IHC: high staining in pachytene spermatocytes). Assess tissue preservation alongside nuclear signal, since damaged morphology complicates cell-level scoring (standard IHC practice). Do not infer a preferred fixative from CDC45’s chromatin association or its reported tissue distribution (UniProt O75419: chromatin association; HPA tissue IHC: nuclear expression).

Where should convincing CDC45 staining appear in an IHC section?

Expect predominantly nuclear staining in CDC45 paraffin-section IHC, with the strongest reported tissue signal in testicular seminiferous ducts (HPA tissue IHC: nuclear expression and testicular abundance). CDC45 associates with chromatin, supporting evaluation of signal within intact nuclei (UniProt O75419: nucleus, chromosome and chromatin association). The subcellular record also reports cytosol and centrosome localisation, so an isolated extranuclear signal needs corroboration rather than automatic dismissal (HPA subcellular: cytosol and centrosome). Compare adjacent cells and a no-primary control before interpreting a faint extranuclear deposit (standard IHC practice). Document whether staining follows nuclear boundaries under the counterstain and whether the same pattern recurs across well-preserved fields (standard IHC practice).

Could epitope choice account for inconsistent CDC45 nuclear staining?

CDC45 has 3 reported isoforms, but the supplied caption does not identify the antibody’s epitope or establish isoform coverage (UniProt O75419: isoforms 1–3; datasheet M01367-2: epitope not stated). Its annotated phosphorylation sites include residues 130, 144 and 148; their effect on this antibody’s staining is unknown (UniProt O75419: modified residues). If sections disagree, check the antibody’s documented immunogen and compare staining under matched retrieval and detection conditions (standard IHC practice). Do not assign an isoform or phosphorylation state from chromogenic intensity alone (standard IHC practice). Report the observed compartment and tissue context alongside the antibody identity (standard IHC practice).

How can IF help assess an ambiguous CDC45 IHC pattern?

Use IF as a separate localisation check when chromogenic CDC45 IHC leaves nuclear boundaries uncertain (standard IF practice). Multiplex CDC45 with a marker identifying the expected cell population, such as pachytene spermatocytes in testis, and inspect overlap cell by cell (HPA tissue IHC: high staining in pachytene spermatocytes; standard IF practice). Choose a fluorophore channel with low tissue autofluorescence and include single-label controls to check bleed-through (standard IF practice). Permeabilise sufficiently to expose the nuclear epitope, since CDC45 is mainly nucleoplasmic and has no annotated transmembrane segment (HPA subcellular: nucleoplasm; UniProt O75419: topology). Treat IF and IHC patterns as complementary observations, documenting any compartment differences (standard IF practice).

What should I check when CDC45 DAB staining looks diffuse?

First compare the section with a no-primary control to locate detection or tissue-derived background (standard IHC practice). The selected caption used 10% goat serum blocking, a biotinylated secondary, a streptavidin-biotin detection complex and DAB (datasheet M01367-2). Check peroxidase blocking and the detection reagents if brown deposit persists without primary antibody (standard chromogenic IHC practice). Reduce nonspecific staining through controlled antibody titration and washing while preserving the expected nuclear signal (standard IHC practice; HPA tissue IHC: nuclear expression). Interpret broad cytoplasmic haze cautiously, because the reported tissue pattern is mainly nuclear even though additional cytosol localisation is described (HPA tissue IHC: nuclear expression; HPA subcellular: cytosol).

How should I quantify CDC45 staining across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION

Define the tissue region and count intact, interpretable nuclei before scoring CDC45 (standard IHC practice; HPA tissue IHC: predominantly nuclear expression). Report the percentage of positive nuclei and an H-score combining the proportions at each intensity level, with the scoring thresholds specified (standard IHC practice). If comparing spatial compartments, report positive-nucleus density per mm² of analysed tissue as a separate measure (standard IHC practice). Normalise counts to eligible nuclei or measured tissue area, and apply identical thresholds across staining runs (standard IHC practice). Record cell type and compartment because high staining in pachytene spermatocytes does not define every cell population in the section (HPA tissue IHC: pachytene spermatocytes high).

How do I distinguish true CDC45 staining from artefact?

Give greatest weight to reproducible nuclear staining in intact cells, consistent with CDC45’s chromatin association and the reported tissue IHC pattern (UniProt O75419: chromatin association; HPA tissue IHC: nuclear expression). Test interpretation against the expected population: pachytene spermatocytes stain highly, while adrenal glandular cells are reported as undetected (HPA tissue IHC: testis and adrenal gland). Examine section edges and necrotic areas separately, since staining there can reflect processing artefact rather than interpretable cell signal (standard IHC practice). A brown deposit in the no-primary control points toward detection background or endogenous enzyme activity (standard chromogenic IHC practice). Document convincing extranuclear staining for further validation rather than assigning it to CDC45 from location alone (HPA subcellular: additional cytosol and centrosome localisation; standard IHC practice).

Boster reagents

Best CDC45 / Cell division control protein 45 homolog IHC Antibodies

The catalog antibody has real IHC data from human ovarian cancer paraffin sections (image caption: M01367-2); the catalog lists human, mouse and rat reactivity (catalog: M01367-2).

Real IHC data

Anti-CDC45L ® Antibody (monoclonal, 6H6)

Cat # M01367-2

View product →

M01367-2 is the sole SKU with a rendered card; its IHC image shows a human ovarian cancer paraffin section (image caption: M01367-2). The catalog lists IHC and human, mouse and rat reactivity for M01367-2 (catalog: M01367-2); no IF image is supplied for it (catalog: M01367-2).

Which to pick: For tissue IHC, choose M01367-2: its mouse monoclonal clone 6H6 has a human ovarian cancer paraffin-section image (catalog and image caption: M01367-2); the caption does not report the fixative (image caption: M01367-2). For IF/ICC, choose rabbit monoclonal clone AD-3, M01367, because IF and ICC are listed applications, although no IF image is supplied (catalog: M01367). For mouse or rat samples, both SKUs list reactivity, but the supplied IHC image for M01367-2 documents human tissue only (catalog: M01367 and M01367-2; image caption: M01367-2).

Each figure is that product's own IHC / IF validation image from its datasheet.

References

UniProt Consortium. UniProt entry O75419 (CDC45_HUMAN, Cell division control protein 45 homolog).
Human Protein Atlas. CDC45 tissue IHC expression (reliability: Enhanced).
Human Protein Atlas. CDC45 subcellular location (ICC-IF): Mainly localized to the nucleoplasm. In addition localized to the cytosol and centrosome..
Human Protein Atlas. CDC45 antibody validation summary (2 antibodies).
Elevated CDC45 Expression Predicts Poorer Overall Survival Prognoses and Worse Immune Responses for Kidney Renal Clear Cell Carcinoma via Single-Cell and Bulk RNA-Sequencing. Biochemical genetics 2024 — PMC11186877.
Significant role of Psf3 expression in non-small-cell lung cancer. Cancer science 2015 — PMC4714687.
Expression of Cell Division Cycle Protein 45 in Tissue Microarrays and the CDC45 Gene by Bioinformatics Analysis in Human Hepatocellular Carcinoma and Patient Outcomes. Medical science monitor : international medical journal of experimental and clinical research 2021 — PMC7919231.
Validation of CDC45 as a novel biomarker for diagnosis and prognosis of gastric cancer. PeerJ 2024 — PMC10956518.
PubMed PMID:9755170 — UniProt-cited evidence.
PubMed PMID:9660782 — UniProt-cited evidence.
PubMed PMID:9724329 — UniProt-cited evidence.