BUB3 / Mitotic checkpoint protein BUB3 · IHC design guide

Design Immunohistochemistry for BUB3

Plan chromogenic BUB3 IHC on paraffin sections using 0.5–1 μg/mL catalog antibody (datasheet: PA1645 IHC-P). Expect widespread nuclear staining, with high signal in colon glandular cells and no detection in liver cholangiocytes (HPA tissue IHC).

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

Immunohistochemistry protocol sheet for BUB3 (IHC for BUB3): expected localisation Nuclear staining in tissue (HPA tissue IHC), antibody PA1645, validated IHC image, and IHC protocol steps — Printable **BUB3** IHC protocol sheet — expected localisation Nuclear staining in tissue (HPA tissue IHC), antibody PA1645, controls and protocol steps. Open the full BUB3 IHC guide →

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BUB3 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	Ubiquitous nuclear staining across cell types (HPA tissue IHC)
	Antigen retrieval	Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen)
	Positive control ⓘ	Adrenal gland+4 more · see all
	Negative control ⓘ	Liver

Important caveats

Reasons your staining may differ from the expected pattern.

	Fixation	Keep fixation consistent (standard IHC practice; not target-specific)
	Caveat	Kinetochore localisation varies with meiotic stage (UniProt)
	Regulation	Expression regulation unreported (UniProt)
	Isoform / epitope	2 isoforms; epitope differences unreported (UniProt)

Section 1

Recommended BUB3 IHC & IF Protocols

Compare the catalog antibody’s IHC-P protocol with two published BUB3 IHC workflows (PMC7190565; PMC8601638).

Recommended immunohistochemistry (IHC-P) protocol parameters
Sample	Tissue sections; selected-image fixative not specified (standard IHC workflow)
Fixation	Image fixative and duration unreported (datasheet PA1645); verify before use.
Sectioning	4–5 µm sections on charged slides (standard)
Deparaffinisation	Xylene, graded ethanol series to water (standard)
Antigen retrieval	Heat-induced epitope retrieval in Tris-EDTA buffer, pH 9.0, 20 min at 95–98 °C (standard rule: nuclear antigen)
Peroxidase block	3% H2O2, 10 min, room temperature (standard)
Blocking	10% normal serum of the secondary host, 30 min, room temperature (standard)
Primary antibody	Rabbit anti-BUB3, 0.5-1μg/ml (datasheet PA1645)
Primary incubation	Overnight at 4 °C (standard)
Detection	HRP-polymer secondary, DAB chromogen 5–10 min (standard)
Counterstain	Hematoxylin, blue, dehydrate and mount (standard)
Expected result	BUB3-positive staining in glandular cells of adrenal gland (HPA tissue IHC: High). HPA tissue profile: Ubiquitous nuclear expression. No signal in the no-primary control.

💡Decision noteStart with Tris-EDTA pH 9.0 at 95–98 °C for 20 min (page retrieval); compare citrate pH 6.0 on matched sections (PMC8601638).

Section 2

What Is the Expected BUB3 Staining Pattern?

BUB3 is primarily nuclear, with centromere and kinetochore localization at defined cell-cycle stages (UniProt O43684: localization). Expect nuclear staining across many tissue cell types, including strong staining in the listed glandular and epithelial cells (HPA: ubiquitous nuclear expression; High in listed cells). HPA rates the tissue IHC pattern Supported, with medium consistency between staining and RNA data (HPA: reliability). BUB3 has no transmembrane segment (UniProt O43684: topology).

What am I looking at on my slide?
Nuclear chromogen in adrenal, colon, or gallbladder glandular cells.	This fits the reported high staining in those cells and the broad nuclear profile (HPA: High in listed glandular cells; ubiquitous nuclear expression). Compare nuclei with neighboring cells and a matched negative control before scoring intensity; the HPA rating is Supported, with medium staining–RNA consistency (HPA: reliability).
Discrete staining associated with chromosomes in a cell undergoing division.	A cell-cycle-specific pattern can fit BUB3 localization to centromeres and kinetochores (UniProt O43684: localization). Evaluate the cell's morphology and counterstain before calling such a focus artefactual. UniProt describes timed kinetochore localization during oocyte meiosis; it does not make every nuclear punctum in a routine tissue section diagnostic of BUB3 (UniProt O43684: localization).
Predominantly membranous or extracellular staining, with little nuclear signal.	This conflicts with the reported nuclear distribution and absence of a transmembrane segment (HPA: ubiquitous nuclear expression; UniProt O43684: topology). Treat the pattern as suspect, then compare with a no-primary control and an independently validated antibody where available (general IHC practice).
Strong staining confined to cholangiocytes, smooth muscle cells, or adipocytes.	Check cell identification and controls before assigning the stain to BUB3: cholangiocytes are Not detected, while smooth muscle cells and adipocytes are Low in the cited HPA tissue profile (HPA: listed cell levels). Cross-reactivity or endogenous detection activity is possible; these HPA observations do not rule out staining in other cells of the same tissue.
Even chromogen across nuclei, cytoplasm, and empty spaces.	This lacks the compartment contrast expected from BUB3's nuclear profile (HPA: ubiquitous nuclear expression). Examine the no-primary control and tissue morphology. Widespread haze may reflect nonspecific binding, incomplete blocking, detection background, or excessive chromogen development (general IHC practice); appearance alone cannot identify which cause applies.

💡Expected BUB3 appearanceCall a result positive when identifiable nuclei stain clearly in a reported high-expression cell population, such as colon glandular cells (HPA: High; ubiquitous nuclear expression); dominant membrane, extracellular, or diffuse field-wide color is suspect rather than the expected BUB3 pattern (UniProt O43684: localization and topology; general IHC practice).

How each factor affects the staining
Tissue and cell selection	Use a reported High population to judge whether the assay can reveal nuclear BUB3; colon glandular cells and cervix squamous epithelial cells are examples (HPA: High). Cholangiocytes are a useful low-signal comparator only in their identified compartment: HPA reports Not detected there, not in every liver cell (HPA: liver cholangiocytes).
Antibody validation	Match the antibody identifier to its application. HPA003601 and CAB037075 have Supported IHC status; HPA071982 has no IHC status in the supplied list (HPA: antibody validation). An Enhanced ICC rating for an antibody should not be substituted for IHC validation (HPA: antibody validation).
Antigen retrieval	No BUB3-specific retrieval condition or fixation-sensitivity result is supplied. For paraffin-section IHC, follow the selected antibody's validated IHC instructions and assess retrieval with positive tissue and a no-primary control (general IHC practice). Changes in signal after retrieval are assay observations, not evidence here of a defined BUB3 fixation effect.
Isoforms and epitope coverage	UniProt lists two BUB3 isoforms and a 1–328 main chain, but the supplied evidence gives no antibody epitope or isoform-specific staining data (UniProt O43684: isoforms and processing). Do not explain a missing stain by isoform exclusion without antibody-specific evidence.
IF/ICC Q: What localization should appear?	A: HPA reports enhanced nucleoplasmic localization in ICC-IF and lists images from A-431, U-251MG, U2OS, and REH (HPA: subcellular). This supports a localization expectation for IF/ICC; it supplies no IF protocol here and does not replace tissue IHC validation (HPA: subcellular; antibody validation).
Detection background	Endogenous enzyme activity and nonspecific secondary-antibody binding can produce chromogenic color independently of the primary antibody (general IHC practice). Use appropriate detection controls and inspect whether color tracks the expected nuclei (HPA: ubiquitous nuclear expression); a colored compartment alone does not prove antigen recognition.

Why is my staining missing, weak or wrong?
Situation	Likely cause	Next action
No nuclear signal in a reported High population.	The assay may lack usable signal, or the sampled cells may have been misidentified; HPA reports High staining in colon glandular cells, for example (HPA: colon). The supplied sources do not identify a BUB3-specific fixation cause.	Verify tissue and cell identity, review the antibody's IHC validation and instructions, and compare the run with a known-positive section processed alongside it (HPA: antibody validation; general IHC practice).
Cytoplasmic or membrane color dominates the section.	That distribution conflicts with nuclear BUB3 and its lack of a transmembrane segment (HPA: ubiquitous nuclear expression; UniProt O43684: topology). Nonspecific binding or detection background may contribute (general IHC practice).	Inspect the no-primary control and counterstained nuclei, then reassess antibody concentration and detection controls according to the assay instructions (general IHC practice). Do not score dominant membrane color as the expected pattern.
Cholangiocytes appear strongly positive.	HPA reports BUB3 as Not detected in cholangiocytes; misidentified cells, cross-reactivity, or endogenous chromogen are possible explanations (HPA: liver cholangiocytes; general IHC practice).	Confirm the cell compartment on the counterstained section and compare no-primary and independent-antibody controls where available (general IHC practice). Avoid extending this cholangiocyte observation to all liver cells.
Every cell and empty area has similar color.	Uniform field-wide color obscures the nuclear contrast expected for BUB3 (HPA: ubiquitous nuclear expression). Background from detection chemistry or excessive development is possible (general IHC practice).	Check the no-primary section, reagent blocking, washes, and chromogen development against the assay instructions (general IHC practice). Score BUB3 only where identifiable nuclei show signal above the local background.
Different antibodies give conflicting nuclear patterns.	Validation is antibody- and application-specific: two listed antibodies are Supported for IHC, while one has no supplied IHC status (HPA: antibody validation). Epitope and isoform coverage cannot be inferred from this payload (UniProt O43684: isoforms).	Record each antibody identifier and its IHC status, compare matched sections and controls, and interpret only reproducible cell-specific nuclear staining (HPA: antibody validation and tissue profile; general IHC practice).
A focal chromosomal signal is called background.	BUB3 can occupy centromeres and kinetochores at defined stages, including the stated interval in oocyte meiosis (UniProt O43684: localization). A focal mark is still insufficient to identify the antigen by itself.	Review cell-cycle morphology, nuclear counterstain, and control sections before classifying the focus (general IHC practice). Report it separately from the broad nuclear tissue pattern described by HPA (HPA: ubiquitous nuclear expression).

Source: UniProt O43684 Human Protein Atlas tissue Human Protein Atlas subcellular

Experiment evidence

IHC & IF images showing BUB3 staining

PA1645 · IHCAnti-Bub3 antibody, PA1645, IHC(P) IHC(P): Rat Testis Tissue

PA1645 · IHCAnti-Bub3 antibody, PA1645, IHC(P) IHC(P): Human Mammary Cancer Tissue

PA1645 · IFAnti-Bub3 antibody, PA1645, ICC ICC: A549 Cell

HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000154473-BUB3/tissue/cerebral+cortex#imid_1331156

HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000154473-BUB3/tissue/colon#imid_1331090

HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000154473-BUB3/tissue/liver#imid_1331075

HPA · PancreasHuman Protein Atlas IHC, Pancreas. Source: https://www.proteinatlas.org/ENSG00000154473-BUB3/tissue/pancreas#imid_1331042

HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA003601; green = BUB3, blue = nucleus, red = microtubules.

← swipe / scroll for more images →

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

Sample controls for BUB3 IHC & IF

🧪Run colon first; its glandular cells should stain (HPA: High in colon glandular cells). Use liver cholangiocytes as the negative tissue (HPA: Not detected in liver cholangiocytes), and compare nearby non-glandular cells on the colon slide as an internal background reference without assuming they are BUB3-negative (HPA: High reported for colon glandular cells).

Positive control tissue: Adrenal gland (Glandular cells, HPA High)

Negative control tissue: Liver (HPA Not detected)

ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show BUB3 in A-431, U-251MG, U2OS, REH, with annotated localisation: Nucleoplasm (enhanced) (HPA subcellular).

Technical controls: Include a no-primary, secondary-only control; an isotype control matched to the primary antibody’s host species and clonality; and BUB3 knockout tissue or a validated peptide-block control (standard IHC practice). For chromogenic IHC, quench endogenous peroxidase and check for endogenous biotin if using avidin–biotin detection; for IF, assess tissue autofluorescence (standard IHC/IF practice).

⚠️Feasibility: A BUB3-specific fixation window and retrieval dependency are unreported in the supplied evidence; optimize antigen retrieval empirically for paraffin IHC (caption: IHC(P) rat testis; fixative unreported). The evidence does not establish whether frozen sections or IF are easier for BUB3, although ICC-IF images show enhanced nucleoplasmic localization (HPA: subcellular). No tissue-specific artefact is reported for colon in the supplied evidence (HPA: High in colon glandular cells).

HPA tissue IHC evidence for BUB3

Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Supported — 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
Adrenal gland	Glandular cells	High	Protein (IHC)	HPA →
Cervix	Squamous epithelial cells	High	Protein (IHC)	HPA →
Colon	Glandular cells	High	Protein (IHC)	HPA →
Fallopian tube	Glandular cells	High	Protein (IHC)	HPA →
Gallbladder	Glandular cells	High	Protein (IHC)	HPA →

Undetected expression · recommended negative controls

Tissue	Cell type	Level	Evidence	Source
Liver	Cholangiocytes	Not detected	Protein (IHC)	HPA →

Section 3

Advanced BUB3 IHC Tips

Troubleshoot BUB3 chromogenic IHC in paraffin sections by checking nuclear staining, retrieval, controls and cell level scoring (UniProt O43684 localisation; HPA tissue IHC).

What retrieval should I start with for weak BUB3 nuclear staining?

Start with Tris-EDTA pH 9.0 heat-induced epitope retrieval at 95–98 °C for 20 min (page retrieval specification). Allow slides to cool in retrieval buffer, then compare nuclear signal and tissue integrity with an otherwise identically processed control section (standard IHC practice; HPA: ubiquitous nuclear expression). If staining remains weak, test an alternative buffer or shorter heating on matched sections as a fallback, recording each condition separately (standard IHC optimisation). Assess glandular cells in colon or adrenal gland as documented high-staining comparators (HPA: High in colon and adrenal gland glandular cells). Judge improvement by clearer nuclear staining without increased diffuse chromogen or damaged morphology (HPA: ubiquitous nuclear expression; standard IHC practice).

Can fixation explain variable BUB3 staining between paraffin sections?

BUB3-specific sensitivity to fixative type or fixation duration is unknown from the supplied evidence (provided BUB3 evidence: no target-specific fixation study). The PA1645 tissue caption identifies rat testis IHC(P) but does not state a fixative (PA1645 tissue-IHC caption). Record each specimen's fixative, fixation interval and processing history, then compare sections processed alike before attributing different nuclear intensity to biology (standard IHC practice; HPA: ubiquitous nuclear expression). If fixation differs between batches, include a shared reference section and evaluate nuclear morphology alongside staining (standard IHC practice). Do not use HPA staining patterns or BUB3 modifications to infer a particular fixation response (HPA: tissue IHC; UniProt O43684: modified residues).

Where should convincing BUB3 chromogen appear in tissue?

Expect predominantly nuclear staining across cell types, with nucleoplasmic localisation supported independently (HPA: ubiquitous nuclear expression; HPA subcellular: nucleoplasm enhanced). BUB3 can also occupy centromere-associated kinetochores during the specified meiotic stages, so focal nuclear puncta require cell-stage context rather than automatic rejection (UniProt O43684: kinetochore localisation from prometaphase I to the metaphase I–anaphase I transition). Use a nuclear counterstain and inspect high magnification fields to separate nuclear chromogen from adjacent cytoplasmic pigment (standard IHC practice). Compare the questioned field with high-staining glandular cells in the same run when available (HPA: High in colon and adrenal gland glandular cells). Diffuse extracellular deposits alone do not match the documented localisation (HPA subcellular: nucleoplasm enhanced; standard IHC practice).

Could isoforms or epitope accessibility change BUB3 IHC staining?

BUB3 has 2 annotated isoforms, while the supplied evidence does not identify which isoforms the catalog antibody recognises (UniProt O43684: isoforms 1 and 2; supplied antibody evidence: epitope unspecified). Its annotated chain spans residues 1–328, with modified residues including acetyllysine 179 and phosphoserine 211 (UniProt O43684: processing and modified residues). Check the antibody's stated immunogen or epitope before interpreting a staining difference as isoform-specific or modification-dependent (standard IHC practice; UniProt O43684: isoforms and modified residues). Compare retrieval conditions on matched sections if nuclear signal is inconsistent, keeping detection settings constant (standard IHC practice). Neither a missing domain annotation nor a modification establishes epitope masking in fixed tissue (UniProt O43684: domains and modified residues).

How should I assess BUB3 in a multiplex IF follow-up?

For a separate IF/ICC experiment, pair BUB3 with a validated marker identifying the expected cell population and use a nuclear counterstain to assess overlap by cell (standard multiplex IF practice; HPA: nucleoplasm enhanced). Choose a fluorophore channel after measuring tissue autofluorescence in an unstained control, especially before assigning a weak nuclear signal (standard IF practice; HPA: ubiquitous nuclear expression). BUB3 has no transmembrane segment and is reported in the nucleus, so optimise permeabilisation for access to an intracellular epitope while monitoring nuclear morphology (UniProt O43684: topology and localisation; standard IF practice). Include single-stain controls to assess channel bleed-through (standard multiplex IF practice). The PA1645 rat testis IHC(P) caption supplies no IF/ICC fixation or multiplex validation (PA1645 tissue-IHC caption).

How can I reduce background without losing BUB3 nuclei?

Compare a no-primary control with the stained section to identify signal arising from detection reagents or endogenous enzyme activity (standard chromogenic IHC practice). Apply a peroxidase block before peroxidase-based DAB detection, and optimise protein blocking and washes according to the detection system (standard chromogenic IHC practice). Titrate the catalog antibody on matched sections rather than assuming a dilution: no working dilution is supplied here (supplied antibody evidence: dilution unspecified; standard IHC optimisation). Inspect edges, folds and necrotic areas separately from intact tissue when judging background (standard IHC practice). Preserve interpretable nuclear signal while reducing diffuse cytoplasmic or extracellular chromogen (HPA: ubiquitous nuclear expression; standard IHC practice).

How should I score heterogeneous BUB3 staining across a section? ⚠ ANSWER MARKED FOR VERIFICATION

Define intact, evaluable regions before scoring and count nuclei within a prespecified cell population rather than pooling distinct compartments (HPA: ubiquitous nuclear expression; standard IHC scoring practice). Report the percentage of positive nuclei and, when intensity is reproducible, an H-score calculated from the percentages at each intensity grade (standard IHC scoring practice). For spatial questions, report positive nuclei per mm² of evaluable tissue and the total nuclei assessed (standard image-analysis practice). Normalise comparisons to the number of eligible nuclei or evaluable area, using the same threshold and chromogen settings across slides (standard IHC scoring practice). Stratify by cell type because HPA records both high and low staining across sampled populations (HPA: tissue IHC).

When is apparent BUB3 positivity likely to be an artefact?

Treat crisp nuclear signal in intact cells as more consistent with BUB3 than isolated extracellular or cytoplasmic deposits (HPA: ubiquitous nuclear expression; HPA subcellular: nucleoplasm enhanced). Check the identified cell type before calling a signal unexpected: high staining is recorded in colon glandular cells, whereas liver cholangiocytes were not detected in the cited survey (HPA: colon glandular cells High; liver cholangiocytes Not detected). Edge staining, necrotic debris and signal persisting without primary antibody warrant investigation as processing or detection artefacts (standard IHC practice). A peroxidase-block control can help test whether endogenous enzyme activity contributes to DAB signal (standard chromogenic IHC practice). Interpret putative kinetochore puncta in their cell-stage context rather than treating every punctum as specific (UniProt O43684: stage-dependent kinetochore localisation).

Boster reagents

Best BUB3 / Mitotic checkpoint protein BUB3 IHC Antibodies

PA1645 has IHC images of rat testis and human mammary cancer paraffin sections, plus ICC images of A549 and HeLa cells (catalog image captions).

Real IHC data Anti-Bub3 antibody, PA1645, IHC(P) IHC(P): Rat Testis Tissue

Anti-Bub3 Antibody ®

Cat # PA1645

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PA1645 is the only SKU with a rendered card; its IHC figure shows rat testis in a paraffin section (card figure caption). Its other captions show human mammary cancer paraffin sections and A549 and HeLa ICC samples (catalog image captions).

Which to pick: Choose PA1645 for tissue IHC because its captions document rat testis and human mammary cancer paraffin sections; the fixative is unreported (PA1645 IHC image captions). For IF/ICC, PA1645 has A549 and HeLa ICC images, while M03118-1 lists ICC/IF without an image in the payload (PA1645 ICC image captions; M03118-1 catalog applications and image alts). Both list human, mouse and rat reactivity; M03118-1 is a rabbit monoclonal, while PA1645 has no clone listed (catalog reactivity and clone fields).

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

References

UniProt Consortium. UniProt entry O43684 (BUB3_HUMAN, Mitotic checkpoint protein BUB3).
Human Protein Atlas. BUB3 tissue IHC expression (reliability: Supported).
Human Protein Atlas. BUB3 subcellular location (ICC-IF): Localized to the nucleoplasm..
Human Protein Atlas. BUB3 antibody validation summary (3 antibodies).
BUBR1 as a Prognostic Biomarker in Canine Oral Squamous Cell Carcinoma. Animals : an open access journal from MDPI 2022 — PMC9687056.
Prognostic value of mitotic checkpoint protein BUB3, cyclin B1, and pituitary tumor-transforming 1 expression in prostate cancer. Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc 2020 — PMC7190565.
Comprehensive analysis of BUBs gene family in lung adenocarcinoma with immunological analysis. Aging 2023 — PMC9970319.
Expression of spindle assembly checkpoint proteins BubR1 and Mad2 expression as potential biomarkers of malignant transformation of oral leukoplakia: an observational cohort study. Medicina oral, patologia oral y cirugia bucal 2021 — PMC8601638.
PubMed PMID:10198256 — UniProt-cited evidence.
PubMed PMID:9660858 — UniProt-cited evidence.
PubMed PMID:14702039 — UniProt-cited evidence.