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MCM5 / DNA replication licensing factor MCM5 · IHC design guide
Design Immunohistochemistry for MCM5
Plan paraffin-section MCM5 IHC around nuclear staining (HPA tissue IHC) and the catalog antibody's IHC validation (datasheet M03642-2). Compare hematopoietic cells with adipocytes, where staining was not detected (HPA tissue IHC), while considering changes in chromatin association during replication (UniProt).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
MCM5 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 tissue (HPA tissue IHC) | |
| Staining pattern | Nuclear staining mainly in hematopoietic and immune cells (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet M03642-2) | |
| Positive control | Appendix+4 more · see all | |
| Negative control | Adipose tissue+4 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent across sections (standard IHC practice; not target-specific). Selected-image fixative and duration unreported (datasheet M03642-2); verify before use. | |
| Caveat | Chromatin association changes as replication progresses (UniProt) | |
| Regulation | Expression regulation is unreported (UniProt) | |
| Isoform / epitope | No isoforms annotated; mature chain spans residues 2–734 (UniProt) |
Section 1
Recommended MCM5 IHC & IF Protocols
The catalog antibody’s IHC-P protocol uses EDTA pH 8.0 retrieval (datasheet: M03642-2). The four published IHC protocols below provide tissue-specific examples (cited PMC methods).
| Sample | Paraffin-embedded human gallbladder adenocarcinoma tissue; fixative not specified (datasheet M03642-2) |
| Fixation | Image fixative and duration unreported (datasheet M03642-2); 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 M03642-2); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet M03642-2) |
| Primary antibody | Mouse monoclonal (clone 4G10) anti-MCM5, 2μg/ml (datasheet M03642-2) |
| Primary incubation | Overnight at 4 °C (datasheet M03642-2) |
| Detection | Streptavidin-biotin complex (SABC), DAB chromogen (datasheet M03642-2) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | MCM5-positive staining in germinal center cells of appendix (HPA tissue IHC: High). HPA tissue profile: Nuclear expression mainly in hematopoietic cells, immune cells, cytotrophoblastic cells in placenta and cells in testis. Also expressed in other cell types. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA pH 8.0 retrieval for the catalog antibody (datasheet: M03642-2); use an article’s retrieval conditions when following that article’s protocol (cited PMC methods).
Section 2
What Is the Expected MCM5 Staining Pattern?
MCM5 should produce predominantly nuclear chromogenic staining in paraffin sections (UniProt P33992: nucleus and chromosome; HPA: tissue IHC). Expect prominent staining in bone marrow hematopoietic cells, lymph node germinal center cells, placental trophoblastic cells, and skin basal layer cells (HPA: High in each). HPA rates its tissue pattern Enhanced, citing consistency between staining and RNA expression (HPA: reliability). MCM5 has no transmembrane segment (UniProt P33992: topology).
| Distinct nuclear staining in hematopoietic or germinal center cells, with little surrounding haze. | This fits the expected compartment and two High tissue patterns (UniProt P33992: nucleus; HPA: High in bone marrow hematopoietic and lymph node germinal center cells). Compare nuclei within each cell population; intensity may differ across populations because HPA reports staining by cell type (HPA: tissue IHC). |
| Predominantly cytoplasmic, membranous, or extracellular chromogen, without convincing nuclear staining. | Treat this as a compartment mismatch and check for artefact before calling it MCM5: UniProt places MCM5 in the nucleus and on chromosomes, while HPA reports nuclear tissue expression (UniProt P33992: subcellular location; HPA: tissue IHC). Diffuse nonnuclear signal alone does not establish target detection (general IHC interpretation). |
| Strong staining in adipocytes or cardiomyocytes, especially with weak expected nuclear staining. | HPA reports MCM5 not detected in those sampled cell populations (HPA: adipose tissue; heart muscle). Assess nonspecific antibody binding or endogenous detection activity using appropriate controls (general chromogenic IHC practice). HPA's result describes those populations; it does not make every cell in either tissue a negative control (HPA: cell-specific tissue IHC). |
| Uniform brown haze across nuclei, cytoplasm, and tissue spaces. | The pattern obscures whether MCM5 is nuclear, so score the slide as uninterpretable until background is reduced (UniProt P33992: nucleus; general IHC interpretation). Compare a no-primary control for detection background and inspect washes and blocking as general IHC checks; HPA does not identify the cause of haze on this slide (general chromogenic IHC practice). |
| No convincing nuclear signal in bone marrow hematopoietic cells or lymph node germinal center cells. | These are High populations in HPA, making absent staining a reason to check the assay before interpreting test specimens (HPA: bone marrow; lymph node). Confirm tissue preservation, antibody use, retrieval conditions, and detection controls as general IHC checks. HPA staining levels do not establish MCM5-specific fixation or retrieval sensitivity (HPA: tissue IHC scope). |
💡Expected MCM5 appearanceCall positive a distinct nuclear chromogenic signal in an HPA High population, such as bone marrow hematopoietic cells; broad cytoplasmic haze or strong staining in HPA not detected adipocytes is a warning pattern, not proof of MCM5 (UniProt P33992: nucleus; HPA: bone marrow and adipose tissue; general IHC interpretation).
| Cell population chosen for comparison | HPA rates germinal center cells in appendix and lymph node, placental trophoblastic cells, and skin basal layer cells High; it rates lung macrophages Low and cardiomyocytes not detected (HPA: tissue IHC). Identify the cell population before comparing intensity across sections (general IHC interpretation). |
| IHC antibody evidence | HPA lists HPA000845 and CAB000101 as IHC Enhanced; its tissue profile also has Enhanced reliability through staining and RNA consistency (HPA: antibodies; reliability). Those ratings support the reported patterns, but they do not specify a dilution, retrieval condition, or performance for an unlisted antibody (HPA: supplied antibody and tissue records). |
| Chromatin association during replication | MCM5 associates with chromatin before nuclei form and detaches as DNA replication progresses (UniProt P33992: subcellular note). This supports nuclear interpretation while allowing biological variation in chromatin association; it does not define a diagnostic chromogen intensity or license a cytoplasmic-positive call (UniProt P33992: subcellular location; general IHC interpretation). |
| Retrieval and molecular features | The supplied record gives no epitope position or MCM5-specific retrieval response (UniProt P33992: supplied record). It reports no transmembrane segment, no signal peptide, 0 glycosylation sites, and a chain spanning residues 2–734 (UniProt P33992: topology and processing). Do not infer fixation sensitivity or a retrieval setting from those features (general IHC interpretation). |
| IF/ICC Q&A: where should signal appear? | HPA reports the nucleoplasm as the enhanced main location in ICC-IF images; UniProt also assigns MCM5 to the nucleus and chromosomes (HPA: subcellular; UniProt P33992: location). This answers the localization question only; IF/ICC staining conditions belong in its separate guide (HPA: supplied subcellular record). |
| Situation | Likely cause | Next action |
|---|---|---|
| Positive tissue has no nuclear chromogen. | An assay failure is possible when bone marrow hematopoietic or lymph node germinal center cells lack their expected High signal (HPA: tissue IHC). The supplied sources do not identify a target-specific technical cause (HPA: supplied records). | Check the primary antibody, detection reagents, and run controls; review the retrieval step against the antibody's own IHC instructions (general chromogenic IHC practice). Do not label retrieval failure as an established MCM5 fixation effect (HPA: tissue IHC scope). |
| Brown signal is mainly cytoplasmic. | That distribution conflicts with nuclear tissue expression and UniProt localization; nonspecific staining or detection artefact is possible (HPA: tissue IHC; UniProt P33992: location; general IHC interpretation). | Compare nuclear counterstain alignment and a no-primary control, then assess blocking and washes before scoring the slide (general chromogenic IHC practice). Require a discrete nuclear pattern in an HPA-positive population (HPA: tissue IHC). |
| Adipocytes or cardiomyocytes stain strongly. | HPA lists those specific populations as not detected; unexpected color could reflect cross-reactivity or endogenous detection activity (HPA: adipose tissue; heart muscle; general chromogenic IHC practice). | Compare a no-primary control and an HPA High cell population on the same run, then review the detection and blocking steps (general IHC practice; HPA: tissue IHC). Interpret other cells in the tissue separately (HPA: cell-specific profile). |
| All compartments show diffuse background. | Broad haze prevents reliable nuclear scoring and may arise from nonspecific binding or detection background (UniProt P33992: nucleus; general chromogenic IHC practice). HPA provides no slide-specific cause (HPA: supplied tissue record). | Inspect no-primary controls, blocking, washes, and detection exposure; repeat scoring only when nuclei can be distinguished from background (general chromogenic IHC practice). |
| Different tissues give different positive-cell fractions. | HPA assigns levels to cell populations: bone marrow hematopoietic cells are High, whereas lung macrophages are Low (HPA: tissue IHC). A whole-section fraction can change with its cellular composition (general IHC interpretation). | Record compartment, intensity, and fraction within each identified cell population; compare like populations and use HPA levels as qualitative references (general IHC scoring; HPA: tissue IHC). |
| An IF/ICC image seems inconsistent with the chromogenic section. | HPA's ICC-IF summary identifies nucleoplasm, while its tissue IHC profile reports nuclear expression across specified cell populations; the records describe different specimen formats (HPA: subcellular; tissue IHC). | Compare nuclear localization, then assess each result against its own HPA record and assay controls (HPA: subcellular and tissue IHC; general assay interpretation). Use the separate IF/ICC guide for that assay's conditions. |
Experiment evidence
IHC & IF images showing MCM5 staining
M03642-2 · IHCIHC analysis of MCM5 using anti-MCM5 antibody (M03642-2). MCM5 was detected in paraffin-embedded section of human gallbladder adenocarcinoma
M03642-2 · IHCIHC analysis of MCM5 using anti-MCM5 antibody (M03642-2). MCM5 was detected in paraffin-embedded section of human lung cancer tissue. Heat m
M03642-2 · IFIF analysis of MCM5 using anti-MCM5 antibody (M03642-2). MCM5 was detected in immunocytochemical section of A431 cells. Enzyme antigen retri
PA1653 · IHCAnti-MCM5 antibody, PA1653, IHC(P) IHC(P): Human Intestinal Cancer Tissue
PA1653 · IHCAnti-MCM5 antibody, PA1653, IHC(P) IHC(P): Human Intestinal Cancer Tissue
PA1653 · IFIF analysis of MCM5 using anti-MCM5 antibody (PA1653). MCM5 was detected in immunocytochemical section of U20S cells. Enzyme antigen retriev
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000100297-MCM5/tissue/cerebral+cortex#imid_259486
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000100297-MCM5/tissue/colon#imid_259396
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000100297-MCM5/tissue/liver#imid_259413
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000100297-MCM5/tissue/kidney#imid_259406
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA052880; green = MCM5, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA052880; green = MCM5, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for MCM5 IHC & IF
🧪Run lymph node first: germinal center cells must show MCM5 staining (HPA: High in lymph node germinal center cells). Use heart muscle cardiomyocytes as the negative tissue (HPA: Not detected in cardiomyocytes); on the positive slide, morphologically identified cells outside germinal centers that lack nuclear staining can serve as internal negative comparators if present (UniProt P33992: nucleus).
Positive control tissue: Appendix (Germinal center cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show MCM5 in A-431, U-251MG, U2OS, U2OS, siRNA 1 (10x), U2OS, siRNA 1 (40x), U2OS, siRNA 2 (10x), U2OS, siRNA 2 (40x), U2OS, scrambled (10x), U2OS, scrambled (40x), NIH 3T3, with annotated localisation: Nucleoplasm (enhanced) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control; a concentration-matched mouse IgG isotype control matched to the primary antibody’s isotype; and MCM5 knockout material as a biological negative if available (caption: mouse anti-MCM5). Block endogenous peroxidase for chromogenic detection and check for endogenous biotin when using the caption’s streptavidin–biotin detection system (caption: SABC with DAB).
⚠️Feasibility: No target-specific fixation window or fixation effect is reported in the supplied evidence, and the selected M03642-2 paraffin-section caption does not state the fixative (caption: fixative unreported). The caption used heat retrieval in EDTA at pH 8.0, but does not establish that retrieval is required; no matched evidence shows that frozen sections or IF are easier (caption: EDTA retrieval; HPA: nucleoplasm in ICC-IF). Dense germinal centers can make individual positive nuclei difficult to score in lymph node sections (HPA: High in germinal center cells).
HPA tissue IHC evidence for MCM5
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — High 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 |
|---|---|---|---|---|
| Appendix | Germinal center cells | High | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | High | Protein (IHC) | HPA → |
| Duodenum | Paneth cells | High | Protein (IHC) | HPA → |
| Endometrium | Glandular cells | High | Protein (IHC) | HPA → |
| Lymph node | Germinal center cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Caudate | Glial cells | Not detected | Protein (IHC) | HPA → |
| Cerebellum | Cells in granular layer | Not detected | Protein (IHC) | HPA → |
| Fallopian tube | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Heart muscle | Cardiomyocytes | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced MCM5 IHC Tips
Troubleshoot MCM5 staining in paraffin sections by checking retrieval, nuclear localisation, detection controls and cell-specific scoring.
What retrieval should I start with if nuclear MCM5 staining is weak?
Start with heat-mediated EDTA pH 8.0 antigen retrieval for paraffin-section MCM5 IHC (datasheet M03642-2). The selected tissue-IHC image used this condition before overnight incubation at 4°C with 2 μg/ml primary antibody (caption M03642-2). If staining remains weak, vary heating time and cooling under controlled conditions, or compare another retrieval buffer as a fallback while holding antibody concentration and detection constant (standard IHC practice). Judge recovery by increased nuclear staining in an expected positive cell population, such as germinal center cells, without a parallel rise in diffuse background (HPA tissue IHC; standard IHC practice).
Could fixation explain weak or uneven MCM5 staining in paraffin sections?
The selected paraffin-section caption does not report a fixative, so target-specific MCM5 fixation sensitivity is unknown (caption M03642-2). Record fixation type and duration for each block, then compare sections processed with the same EDTA pH 8.0 retrieval and detection conditions (datasheet M03642-2; standard IHC practice). Check whether nuclear staining varies across the section or between similarly stained control blocks before changing the primary antibody concentration (standard IHC practice). HPA tissue patterns establish useful positive cell populations, but they do not establish which fixation conditions preserve this antibody’s epitope (HPA tissue IHC; caption M03642-2).
How should I assess cytoplasmic or absent staining when MCM5 is expected in nuclei?
Prioritise nuclear staining: MCM5 is annotated in the nucleus and on chromosomes, while HPA reports enhanced nucleoplasmic localisation (UniProt P33992 subcellular; HPA subcellular). Chromatin association changes as DNA replication progresses, so nuclear signal need not look identical in every cell (UniProt P33992 subcellular). If staining appears predominantly cytoplasmic, inspect the hematoxylin counterstain, section integrity and nonspecific background before scoring those cells as positive (standard IHC practice). Compare the suspect area with expected nuclear-positive populations, including germinal center cells or hematopoietic cells, using the same staining run and exposure conditions (HPA tissue IHC; standard IHC practice).
Could isoforms or epitope masking account for inconsistent MCM5 IHC?
The supplied UniProt record lists 0 isoforms, so an isoform-specific explanation lacks support from this record (UniProt P33992 isoforms). MCM5 has no transmembrane segment, and its MCM C-terminal AAA(+) ATPase domain spans residues 314–654 (UniProt P33992 topology; UniProt P33992 domains). Reported modifications include phosphoserine at 315 and 605 and acetyllysine at 392 and 396, but their effect on this antibody’s staining is unreported (UniProt P33992 modified residues; caption M03642-2). Check the antibody’s stated immunogen or epitope if available, then test retrieval changes against a fixed positive control rather than attributing variable staining to a modification (standard IHC practice).
How can I use IF to investigate an ambiguous chromogenic MCM5 pattern?
Use IF as a separate localisation check while keeping paraffin-section chromogenic IHC as the primary readout (standard IHC/IF practice). Pair MCM5 with a validated marker for the expected cell population, such as germinal center cells, and check that signal lies within the same nuclei (HPA tissue IHC; standard IF practice). Choose a fluorophore channel with low sample autofluorescence and include single-channel controls to distinguish bleed-through from colocalisation (standard IF practice). Because MCM5 is nucleoplasmic and has no transmembrane segment, optimise permeabilisation for access to the nuclear epitope; the supplied IHC caption does not validate an IF fixation or permeabilisation condition (HPA subcellular; UniProt P33992 topology; caption M03642-2).
What should I check when MCM5 DAB staining spreads beyond nuclei?
The selected chromogenic workflow used 10% goat serum blocking, 2 μg/ml primary antibody, a biotinylated secondary and DAB development (caption M03642-2). For diffuse signal, compare a no-primary control and review blocking, washes, antibody concentration and DAB development time on adjacent sections (standard IHC practice). Include an endogenous peroxidase block as a general chromogenic IHC step, and assess endogenous biotin if background tracks tissue structures in this biotin-based workflow (standard IHC practice; caption M03642-2). Score MCM5 only where staining resolves to nuclei; widespread cytoplasmic colour conflicts with its reported nuclear and nucleoplasmic localisation (UniProt P33992 subcellular; HPA subcellular).
How should I quantify MCM5-positive cells across heterogeneous tissue sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and region before scoring, then record the percentage of nuclei above a fixed staining threshold (standard IHC practice). Where intensity matters, calculate a nuclear H-score from the percentages of cells in intensity categories 0–3, yielding a 0–300 scale (standard IHC practice). Alternatively, count positive nuclei per mm², and normalise that density to the sampled tissue area and relevant cell population (standard IHC practice). Keep retrieval, DAB development, counterstain and threshold settings consistent across sections; report cell-specific results because HPA shows high MCM5 in some cell populations and undetected staining in others (HPA tissue IHC; standard IHC practice).
How can I distinguish genuine MCM5 positivity from staining artefact?
A credible MCM5-positive cell has nuclear staining consistent with UniProt’s nuclear assignment and HPA’s nucleoplasmic localisation (UniProt P33992 subcellular; HPA subcellular). Compare the result with expected positive populations, such as hematopoietic or germinal center cells, and with cell populations reported as not detected, such as adipocytes (HPA tissue IHC). Treat staining confined to section edges, necrotic areas or sites of endogenous enzyme activity cautiously, and inspect a no-primary control before assigning positivity (standard IHC practice). Predominantly cytoplasmic colour or an indiscriminate signal across cell types warrants review of retrieval, blocking and detection before biological interpretation (UniProt P33992 subcellular; HPA tissue IHC; standard IHC practice).
Boster reagents
Best MCM5 / DNA replication licensing factor MCM5 IHC Antibodies
Anti-MCM5 antibodies have IHC images from human paraffin sections (catalog IHC captions) and IF/ICC images from A431 and U20S cells (catalog IF captions). Both list human, mouse and rat reactivity (catalog applications).
Real IHC data
Anti-MCM5 ® Antibody (monoclonal, 4G10)
Cat # M03642-2
Real IHC data
Anti-MCM5 Antibody ®
Cat # PA1653
M03642-2 has IHC images from human gallbladder adenocarcinoma, lung cancer and pancreatic cancer paraffin sections, plus an A431 IF/ICC image (M03642-2 image captions). PA1653 has a human intestinal cancer IHC image and a U20S IF/ICC image (PA1653 image captions).
Which to pick: For tissue IHC, M03642-2 offers a mouse monoclonal clone 4G10 with documented EDTA pH 8.0 retrieval and 2 μg/ml incubation in a paraffin section; the fixative is unreported (catalog clone field; M03642-2 IHC caption). For IF/ICC, PA1653 offers a rabbit host and a documented 2 μg/ml U20S cell image (catalog host field; PA1653 IF caption). For cross-species planning, both list human, mouse and rat reactivity, while their supplied IHC images show human samples (catalog reactivity fields; M03642-2 and PA1653 IHC captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P33992 (MCM5_HUMAN, DNA replication licensing factor MCM5).
- Human Protein Atlas. MCM5 tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. MCM5 subcellular location (ICC-IF): Localized to the nucleoplasm..
- Human Protein Atlas. MCM5 antibody validation summary (3 antibodies).
- Minichromosome maintenance protein 5 - a promising prognostic marker of oral epithelial dysplasias and oral squamous cell carcinomas. Journal of oral and maxillofacial pathology : JOMFP 2023 — PMC10829438.
- MCMs expression in lung cancer: implication of prognostic significance. Journal of Cancer 2017 — PMC5688916.
- Diagnosis of pancreaticobiliary malignancy by detection of minichromosome maintenance protein 5 in bile aspirates. British journal of cancer 2008 — PMC2391096.
- MCM5 Aggravates the HDAC1-Mediated Malignant Progression of Lung Cancer. Frontiers in cell and developmental biology 2021 — PMC8366603.
- PubMed PMID:15461802 — UniProt-cited evidence.
- PubMed PMID:10591208 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for MCM5 (DNA replication licensing factor MCM5). Expected localisation Nuclear staining in tissue (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet M03642-2). Validated IHC antibody: M03642-2 at 2μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Appendix (Germinal center cells); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human gallbladder adenocarcinoma tissue; fixative not specified (datasheet M03642-2); 2. Fixation: Image fixative and duration unreported (datasheet M03642-2); 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 M03642-2); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet M03642-2); 8. Primary antibody: Mouse monoclonal (clone 4G10) anti-MCM5, 2μg/ml (datasheet M03642-2); 9. Primary incubation: Overnight at 4 °C (datasheet M03642-2). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific). Selected-image fixative and duration unreported (datasheet M03642-2); verify before use.; Caveat: Chromatin association changes as replication progresses (UniProt); Isoform / epitope: No isoforms annotated; mature chain spans residues 2–734 (UniProt); Regulation: Expression regulation is unreported (UniProt). Evidence: UniProt P33992, HPA tissue IHC · Enhanced, HPA ICC-IF · nucleoplasm., Datasheet M03642-2, PMC10829438, PMC5688916. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/MCM5/immunohistochemistry-ihc-guide.