CBX3 / Chromobox protein homolog 3 · IHC design guide

Design Immunohistochemistry for CBX3

Plan CBX3 IHC in paraffin sections around its widespread nuclear staining (HPA tissue IHC). The guide uses high-staining alveolar type I cells as a reference (HPA tissue IHC) and the catalog antibody’s IHC-P conditions (datasheet A01142).

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

Immunohistochemistry protocol sheet for CBX3 (IHC for CBX3): expected localisation Predominantly nuclear (HPA tissue IHC), antibody A01142, validated IHC image, and IHC protocol steps — Printable **CBX3** IHC protocol sheet — expected localisation Predominantly nuclear (HPA tissue IHC), antibody A01142, controls and protocol steps. Open the full CBX3 IHC guide →

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CBX3 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	Predominantly nuclear (HPA tissue IHC)
	Staining pattern	Nuclear staining across many tissue cell types (HPA tissue IHC)
	Antigen retrieval	Citrate pH 6 HIER, heat-mediated (datasheet A01142)
	Positive control ⓘ	Lung+4 more · see all
	Negative control ⓘ	None in HPA (detected in all 44 tissues); use no-primary + isotype controls

Important caveats

Reasons your staining may differ from the expected pattern.

	Fixation	Keep fixation conditions consistent across sections. (standard IHC practice; not target-specific)
	Caveat	Widespread normal-cell staining limits tumor specificity (HPA tissue IHC)
	Regulation	Low tissue specificity (HPA tissue RNA)
	Isoform / epitope	No listed isoforms; no membrane-sided epitope distinction (UniProt)

Section 1

Recommended CBX3 IHC & IF Protocols

The catalog antibody’s IHC-P protocol uses heat-mediated citrate retrieval at pH 6 (datasheet A01142). The published CBX3 protocols below cover brain, tongue and kidney tissues (PMC6540543; PMC5920882; PMC8357267).

Recommended immunohistochemistry (IHC-P) protocol parameters
Sample	Paraffin-embedded human mammary cancer tissue; fixative not specified (datasheet A01142)
Fixation	Image fixative and duration unreported (datasheet A01142); verify before use.
Sectioning	4–5 µm sections on charged slides (standard)
Deparaffinisation	Xylene, graded ethanol series to water (standard)
Antigen retrieval	Heat retrieval: Citrate pH 6, 20 min (datasheet A01142)
Peroxidase block	3% H2O2, 10 min, room temperature (standard)
Blocking	10% goat serum (datasheet A01142)
Primary antibody	Rabbit anti-CBX3, 0.5-1μg/ml (datasheet A01142)
Primary incubation	Overnight at 4 °C (datasheet A01142)
Detection	Streptavidin-biotin complex (SABC), DAB chromogen (datasheet A01142)
Counterstain	Hematoxylin, blue, dehydrate and mount (standard)
Expected result	CBX3-positive staining in alveolar cells type I of lung (HPA tissue IHC: High). HPA tissue profile: Ubiquitous nuclear expression. No signal in the no-primary control.

💡Decision noteStart with heat-mediated citrate retrieval at pH 6 (datasheet A01142). For a published starting condition, one study boiled sections in 10 mM citrate for 15 min (PMC5920882).

Section 2

What Is the Expected CBX3 Staining Pattern?

CBX3 is a nuclear protein with no transmembrane segment (UniProt Q13185: location and topology). In paraffin-section IHC, expect nuclear staining across many cell types: HPA describes ubiquitous nuclear expression and low tissue specificity, with high staining in selected cells (HPA: tissue IHC). HPA rates the tissue pattern Supported because antibody staining and RNA expression are consistent (HPA: tissue IHC).

What am I looking at on my slide?
Nuclear chromogenic signal in alveolar type I cells, cytotrophoblasts, melanocytes, or pachytene spermatocytes.	This fits the expected compartment and HPA's High staining calls for those cells (UniProt Q13185: nucleus; HPA: tissue IHC). Score the relevant cells and their nuclei, rather than treating every cell in the section as equivalent (general IHC practice).
Predominantly cytoplasmic, membranous, or extracellular staining with little nuclear signal.	This conflicts with the expected nuclear location and lack of a transmembrane segment (UniProt Q13185: location and topology). Treat it as a possible artefact; review morphology, detection controls, and antibody validation before interpreting it as CBX3 (general IHC practice).
Strong signal in cells HPA lists as Low, such as hepatocytes or skeletal myocytes.	Investigate cross-reactivity or endogenous detection activity, especially if staining is outside nuclei (HPA: Low in hepatocytes and myocytes; UniProt Q13185: nucleus). Low is not negative: HPA reports ubiquitous nuclear expression, so weak nuclear staining alone is plausible (HPA: tissue IHC).
Broad haze or deposit across nuclei, cytoplasm, and empty tissue spaces.	Poorly localized staining does not match the nuclear reference pattern (HPA: tissue IHC; UniProt Q13185: nucleus). Compare a negative reagent control and examine wash, blocking, and chromogen conditions as general IHC checks; the supplied sources do not identify a CBX3-specific cause.
No nuclear signal in a section containing a documented High cell population.	This is unexpected for that cell population (HPA: High in alveolar type I cells, cytotrophoblasts, melanocytes, and pachytene spermatocytes). Confirm that the relevant cells are present and preserved, then check assay controls and IHC conditions; absence alone does not establish absent CBX3 (general IHC practice).

💡Expected CBX3 appearanceCall positive when staining is predominantly nuclear in the relevant cells, with strong signal plausible in HPA High populations; isolated cytoplasmic or diffuse background signal without matching nuclei is suspect (UniProt Q13185: nucleus; HPA: tissue IHC).

How each factor affects the staining
Nuclear distribution	UniProt places CBX3 in the nucleus, associated mainly with euchromatin and largely excluded from constitutive heterochromatin (UniProt Q13185: location). Interpret nuclear staining by its distribution; the record does not require a uniform nuclear fill.
Tissue and cell context	HPA calls expression ubiquitous but assigns different staining levels to named cells, including High alveolar type I cells and Low hepatocytes (HPA: tissue IHC). Use the cell identity and compartment together; the Low list supplies weaker comparators, not negative controls.
Antibody evidence	Both listed antibodies have Supported IHC status; HPA004902 also has Supported ICC status (HPA: antibody validation). HPA's overall tissue reliability is Supported, based on staining and RNA consistency (HPA: tissue IHC). These labels support comparison with the reference pattern, not specificity of every deposit.
Topology and processing	The record has no transmembrane segment or signal peptide and lists no propeptide (UniProt Q13185: topology and processing). A dominant membrane or secreted-looking pattern therefore lacks support from this record; verify it before assigning it to CBX3.
IF/ICC Q: What location should I expect?	A: Mainly nucleoplasmic signal, with additional nuclear bodies, is supported in HPA ICC-IF (HPA: subcellular). This is a location cross-check only; the IF/ICC guide carries its own method, and no IF/ICC protocol option is supplied here.

Why is my staining missing, weak or wrong?
Situation	Likely cause	Next action
High-background chromogen obscures nuclei.	Nonspecific detection or endogenous enzyme activity is possible (general IHC practice); the sources give no CBX3-specific background mechanism.	Inspect a negative reagent control, confirm endogenous activity blocking when relevant, and adjust wash or detection conditions (general IHC practice). Reassess nuclear localization against HPA's ubiquitous nuclear pattern (HPA: tissue IHC).
Only cytoplasm or cell borders stain.	That compartment disagrees with CBX3's nuclear location and no-transmembrane topology (UniProt Q13185: location and topology).	Check section morphology and controls, then compare another IHC-validated antibody if available; the supplied HPA list contains two Supported IHC antibodies (HPA: antibody validation).
A supposed negative comparator shows faint nuclear signal.	HPA lists no negative cell populations and describes ubiquitous nuclear expression; its Low calls do not mean absent protein (HPA: tissue IHC).	Use a negative reagent control to assess detection background (general IHC practice). Compare cell-specific intensity and nuclear localization with HPA's High and Low examples (HPA: tissue IHC).
No signal appears in a documented High population.	An assay or tissue-quality problem is possible; HPA reports High staining in specific populations, but the supplied sources do not establish CBX3 fixation sensitivity (HPA: tissue IHC).	Verify that the named cells are present, run an appropriate positive control, and review retrieval and detection settings as general IHC checks. Do not infer a CBX3-specific retrieval requirement (general IHC practice).
Nuclear staining is patchy within an otherwise positive section.	Cell composition may vary, and HPA reports different staining levels among cell types (HPA: tissue IHC). Uneven staining can also arise from section or detection conditions (general IHC practice).	Map staining to identified cell types and inspect section integrity and control staining before assigning a biological pattern (general IHC practice).
A candidate nuclear body pattern is seen in IHC.	HPA supports additional nuclear-body localization in ICC-IF, but that observation does not by itself validate discrete bodies in paraffin-section IHC (HPA: subcellular).	First confirm predominant nuclear staining and adequate controls. Record bodies as an observation, and use the separate IF/ICC guide if subnuclear localization needs assessment (UniProt Q13185: nucleus; HPA: subcellular).

Source: UniProt Q13185 Human Protein Atlas tissue Human Protein Atlas subcellular

Experiment evidence

IHC & IF images showing CBX3 staining

IHC analysis of HP1 gamma using anti-HP1 gamma antibody (A01142). HP1 gamma was detected in paraffin-embedded section of human mammary cancer tissue. Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1μg/ml rabbit anti-HP1 gamma Antibody (A01142) overnight at 4°C. Biotinylated goat anti-rabbit 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 # SA1022) with DAB as the chromogen.

A01142 · IHCIHC analysis of HP1 gamma using anti-HP1 gamma antibody (A01142). HP1 gamma was detected in paraffin-embedded section of human mammary cance

IHC analysis of HP1 gamma using anti-HP1 gamma antibody (A01142). HP1 gamma was detected in paraffin-embedded section of human rectal cancer tissue. Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1μg/ml rabbit anti-HP1 gamma Antibody (A01142) overnight at 4°C. Biotinylated goat anti-rabbit 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 # SA1022) with DAB as the chromogen.

A01142 · IHCIHC analysis of HP1 gamma using anti-HP1 gamma antibody (A01142). HP1 gamma was detected in paraffin-embedded section of human rectal cancer

IF analysis of HP1 gamma using anti-HP1 gamma antibody (A01142). HP1 gamma was detected in immunocytochemical section of U20S cell. 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 2μg/mL rabbit anti-HP1 gamma Antibody (A01142) overnight at 4°C. DyLight®488 Conjugated Goat Anti-Rabbit IgG (BA1127) was used as secondary antibody at 1:100 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.

A01142 · IFIF analysis of HP1 gamma using anti-HP1 gamma antibody (A01142). HP1 gamma was detected in immunocytochemical section of U20S cell. Enzyme a

HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000122565-CBX3/tissue/cerebral+cortex#imid_1852600

HPA · Thyroid glandHuman Protein Atlas IHC, Thyroid gland. Source: https://www.proteinatlas.org/ENSG00000122565-CBX3/tissue/thyroid+gland#imid_1852552

HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000122565-CBX3/tissue/colon#imid_1852518

HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000122565-CBX3/tissue/liver#imid_1852519

HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA004902; green = CBX3, blue = nucleus, red = microtubules.

← swipe / scroll for more images →

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

Sample controls for CBX3 IHC & IF

🧪Run lung first: alveolar type I cells should show nuclear staining (HPA: High in alveolar cells type I; UniProt Q13185: nuclear localization). HPA detects CBX3 in all 44 scored tissues, so there is no supported negative tissue; use no-primary and isotype controls, and expect cells without specific signal on the positive slide to show only background staining rather than treating any cell type as a validated internal negative (HPA: no negative rows; standard IHC practice).

Positive control tissue: Lung (Alveolar cells type I, HPA High)

Negative control tissue: None in HPA: CBX3 is detected in all 44 scored tissues. Use a no-primary (secondary-only) and an isotype control instead.

ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show CBX3 in A-431, U-251MG, U2OS, with annotated localisation: Nucleoplasm (supported) (HPA subcellular).

Technical controls: Include a no-primary, secondary-only section, a concentration-matched nonimmune rabbit IgG isotype control, and CBX3 knockout tissue or cells as a biological negative (caption: rabbit primary antibody; standard IHC practice). Block endogenous peroxidase and check for endogenous biotin in lung sections because the reported detection uses a biotinylated secondary and DAB (caption: biotinylated secondary, SABC and DAB; standard IHC practice).

⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected A01142 paraffin-section caption does not state the fixative (caption: fixative unreported). The caption reports heat retrieval in citrate buffer at pH 6 for 20 minutes; this is a starting condition, not evidence that retrieval is required, and the supplied evidence does not establish whether frozen sections or IF are easier (caption: retrieval conditions; HPA: ICC-IF images). In lung, endogenous peroxidase can create chromogenic background, so interpret nuclear DAB only after reviewing the negative controls (standard IHC practice; UniProt Q13185: nuclear localization).

HPA tissue IHC evidence for CBX3

Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Supported — 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
Lung	Alveolar cells type I	High	Protein (IHC)	HPA →
Placenta	Cytotrophoblasts	High	Protein (IHC)	HPA →
Skin	Melanocytes	High	Protein (IHC)	HPA →
Testis	Pachytene spermatocytes	High	Protein (IHC)	HPA →
Adipose tissue	Adipocytes	Medium	Protein (IHC)	HPA →

Undetected expression · recommended negative controls

Tissue	Cell type	Level	Evidence	Source
None in HPA: CBX3 is detected in all 44 scored tissues. Use a no-primary (secondary-only) and an isotype control instead.

Section 3

Advanced CBX3 IHC Tips

Use nuclear localisation and the catalog antibody’s paraffin-section IHC conditions to troubleshoot CBX3 staining, then score only interpretable cells (UniProt Q13185; datasheet A01142).

Which retrieval conditions should I try first for CBX3 in paraffin sections?

Use heat-mediated citrate buffer at pH 6 for 20 minutes as the starting retrieval condition (datasheet A01142). The catalog antibody produced a chromogenic signal in a paraffin section after this treatment, followed by 1 μg/mL primary antibody overnight at 4°C (datasheet A01142). If nuclear staining is weak, compare a modest change in heating time on adjacent sections while keeping antibody concentration and detection constant (standard IHC practice). Judge improvement by crisp nuclear staining with limited extranuclear signal, since CBX3 is predominantly nuclear (UniProt Q13185; HPA subcellular). Record retrieval conditions for every comparison, because excess heating can damage section morphology (standard IHC practice).

How should I troubleshoot fixation-related loss of CBX3 staining?

Target-specific fixation sensitivity is unknown: the selected paraffin-section caption does not state a fixative (datasheet A01142). Record the actual fixative, fixation duration, tissue thickness and processing history before comparing a weak case with a well-stained control (standard IHC practice). Run sections processed under comparable conditions together, using the documented citrate pH 6, 20-minute retrieval as a starting point (datasheet A01142; standard IHC practice). If only one processing batch fails, compare its nuclear morphology and staining with a separately processed control before changing antibody concentration (standard IHC practice). Do not assign the loss specifically to fixation without that comparison (standard IHC practice).

Where should convincing CBX3 staining appear in tissue sections?

Expect predominantly nuclear staining: tissue IHC shows a ubiquitous nuclear profile, and subcellular data place CBX3 mainly in the nucleoplasm, with additional nuclear-body localisation (HPA tissue IHC; HPA subcellular). CBX3 associates with euchromatin and is largely excluded from constitutive heterochromatin, so assess nuclear distribution rather than requiring uniform chromatin staining (UniProt Q13185). Compare DAB signal with a nuclear counterstain and inspect several intact fields before calling a cell positive (standard IHC practice). Predominantly membrane-restricted staining is discordant with the reported localisation and absence of a transmembrane segment (UniProt Q13185). Mitotic-pole association is listed as potential, so isolated mitotic staining needs cautious interpretation (UniProt Q13185).

Could isoforms or epitope masking explain discordant CBX3 results?

The supplied record lists 0 isoforms, so an isoform-specific explanation is unsupported here (UniProt Q13185). CBX3 has a chromo domain at residues 30–88, a chromoshadow domain at 121–179, and annotated modified residues, but the catalog antibody’s epitope is not specified in this payload (UniProt Q13185; datasheet A01142). Do not claim that a particular modification blocks binding without epitope mapping and a direct comparison (standard IHC interpretation). Instead, compare adjacent sections across controlled retrieval conditions and include the documented positive staining workflow (datasheet A01142; standard IHC practice). Keep the tissue region, detection chemistry and exposure to chromogen comparable when judging a change (standard IHC practice).

How can I check CBX3 localisation by multiplex IF alongside IHC?

For IF, pair CBX3 with a marker identifying the cell population of interest and a nuclear counterstain, then compare cellular assignment with the chromogenic section (standard IF practice). CBX3 is chiefly nucleoplasmic, and the record reports no transmembrane segment, so permeabilise sufficiently for antibody access to the nuclear antigen (HPA subcellular; UniProt Q13185; standard IF practice). Choose a fluorophore channel with low tissue autofluorescence and inspect a no-primary control before interpreting faint signal (standard IF practice). Acquire each channel separately to check spectral bleed-through, then score nuclear overlap within the marker-defined cells (standard IF practice). The paraffin IHC caption supplies no IF fixation or permeabilisation conditions (datasheet A01142).

How do I reduce diffuse or extranuclear DAB background?

First compare a no-primary section with the stained section to separate detection-system background from antibody-associated staining (standard IHC practice). The documented workflow used 10% goat serum blocking, 1 μg/mL primary antibody overnight at 4°C, a biotinylated secondary antibody and DAB detection (datasheet A01142). For diffuse signal, check serum block, washing, primary concentration and DAB development time one variable at a time (standard IHC practice). Include a peroxidase block when using peroxidase-based chromogenic detection, and evaluate endogenous biotin if background persists with a biotin-based system (standard IHC practice). Prioritise sharply localised nuclear staining over diffuse cytoplasmic colour when interpreting CBX3 (HPA tissue IHC; standard IHC practice).

What is a defensible way to quantify CBX3 IHC staining? ⚠ ANSWER MARKED FOR VERIFICATION

Define the cell population and tissue region before scoring, then count only intact nuclei with interpretable chromogen and counterstain (standard IHC practice; HPA tissue IHC). Report the percentage of nuclear-positive cells and, when intensity grading is reproducible, an H-score from intensity categories 0–3 (standard IHC practice). Normalise counts to all evaluable nuclei in the selected population, or report positive-cell density per mm² of viable tissue when spatial abundance is the question (standard IHC practice). Apply the same threshold, retrieval and DAB development conditions across sections (standard IHC practice). Record cell type because HPA reports high staining in some populations and low staining in others (HPA tissue IHC).

How can I distinguish true CBX3 positivity from staining artefacts?

A credible positive signal lies in intact nuclei, consistent with the ubiquitous nuclear tissue profile and supported nucleoplasmic localisation (HPA tissue IHC; HPA subcellular). Compare staining with expected cell populations: HPA reports high signal in alveolar type I cells and cytotrophoblasts, but low signal in hepatocytes and skeletal myocytes (HPA tissue IHC). Examine section edges and necrotic areas separately, since uneven processing or trapped chromogen can create misleading colour there (standard IHC practice). Use a no-primary control to assess endogenous enzyme and detection background, particularly if colour appears outside nuclei (standard IHC practice). Do not treat a wrong-compartment signal alone as evidence of CBX3 expression (UniProt Q13185; standard IHC interpretation).

Boster reagents

Best CBX3 / Chromobox protein homolog 3 IHC Antibodies

A01142 has pictured IHC in human mammary and rectal cancer paraffin sections and mouse and rat small intestine paraffin sections (catalog IHC captions), plus ICC/IF in U20S cells (catalog IF caption).

Real IHC data

Anti-HP1 gamma/CBX3 Antibody ®

Cat # A01142

View product →

A01142 is the SKU that will render; its IHC captions show paraffin sections from human mammary and rectal cancers and mouse and rat small intestine (catalog IHC captions). Its IF caption shows staining in U20S cells (catalog IF caption).

Which to pick: Choose A01142 for paraffin-section IHC because its own captions document those samples (catalog IHC captions); the fixative is unreported (catalog IHC captions). Choose A01142 for IF/ICC because those applications and a cell image are listed (catalog applications; catalog IF caption). For IHC across human, mouse and rat, A01142 has pictured examples in all three species (catalog IHC captions); M01142-1 is a rabbit monoclonal listed for IHC in those species, but has no IHC image in the payload (catalog applications and reactivity; catalog IHC image alts).

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

References

UniProt Consortium. UniProt entry Q13185 (CBX3_HUMAN, Chromobox protein homolog 3).
Human Protein Atlas. CBX3 tissue IHC expression (reliability: Supported).
Human Protein Atlas. CBX3 subcellular location (ICC-IF): Mainly localized to the nucleoplasm. In addition localized to the nuclear bodies..
Human Protein Atlas. CBX3 antibody validation summary (2 antibodies).
Integrated analysis of 34 microarray datasets reveals CBX3 as a diagnostic and prognostic biomarker in glioblastoma. Journal of translational medicine 2019 — PMC6540543.
CBX3/HP1γ is upregulated in tongue squamous cell carcinoma and is associated with an unfavorable prognosis. Experimental and therapeutic medicine 2018 — PMC5920882.
CBX3 Promotes Gastric Cancer Progression and Affects Factors Related to Immunotherapeutic Responses. Cancer management and research 2020 — PMC7569062.
Comprehensive Analysis of the Expression and Prognosis Value of Chromobox Family Members in Clear Cell Renal Cell Carcinoma. Frontiers in oncology 2021 — PMC8357267.
PubMed PMID:8663349 — UniProt-cited evidence.
PubMed PMID:10664448 — UniProt-cited evidence.
PubMed PMID:15489334 — UniProt-cited evidence.