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CBX3 / Chromobox protein homolog 3 · IHC design guide
Design Immunohistochemistry for CBX3
Plan paraffin-section CBX3 IHC around its ubiquitous nuclear staining pattern (HPA tissue IHC). Use the catalog antibody’s 0.5–1 μg/mL IHC dilution as a starting range (datasheet A01142), and compare nuclear staining across cells within each section (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
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 | Nuclei across tissues (HPA tissue IHC) | |
| Staining pattern | Ubiquitous nuclear staining across 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 | Ubiquitous nuclear signal limits unstained-cell controls (HPA tissue IHC) | |
| Regulation | No expression regulator annotated (UniProt) | |
| Isoform / epitope | No isoforms annotated; chain spans residues 2–183 (UniProt) |
Section 1
Recommended CBX3 IHC & IF Protocols
The catalog antibody’s IHC protocol (datasheet A01142) is accompanied by published CBX3 tissue protocols (PMC6540543; PMC5920882; PMC8357267).
| 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 retrieval in citrate buffer at pH 6 (datasheet A01142; PMC5920882), then assess nuclear staining (UniProt Q13185).
Section 2
What Is the Expected CBX3 Staining Pattern?
CBX3 should appear predominantly in nuclei across many cell types, consistent with the HPA description of ubiquitous nuclear expression and supported tissue IHC reliability (HPA: tissue IHC). It is a nuclear protein with no transmembrane segment (UniProt Q13185: subcellular location and topology). For a clear positive reference, examine alveolar type I cells, cytotrophoblasts, melanocytes, or pachytene spermatocytes, each reported with high staining (HPA: tissue IHC).
| Distinct nuclear chromogen in an expected cell population, with interpretable tissue structure and limited staining outside nuclei. | This fits CBX3 localisation (UniProt Q13185: nucleus; HPA: ubiquitous nuclear expression). Compare intensity within identified cells: HPA reports high staining in lung alveolar type I cells and placental cytotrophoblasts, but low staining in liver hepatocytes. Those categories describe observed patterns, not universal cutoffs for every section (HPA: tissue IHC). |
| Predominantly cytoplasmic, membranous, or extracellular deposit while the expected nuclei remain unstained. | That compartment conflicts with the supported nuclear pattern (UniProt Q13185: nucleus; HPA: tissue IHC). Treat it as a suspect IHC result and check morphology and detection controls (general IHC practice). UniProt notes possible association with microtubules and mitotic poles during mitosis; that potential does not establish diffuse cytoplasmic staining as the expected tissue pattern (UniProt Q13185: subcellular location). |
| Strong staining appears selectively in an unexpected cell population, while an expected high-staining population is weak or absent. | Check cell identification and controls before interpreting the selective signal; cross-reactivity or endogenous detection activity are possibilities (general IHC practice). CBX3 is described as ubiquitously nuclear, so signal in another cell type alone does not prove a false positive, and the supplied HPA profile provides no negative tissue reference (HPA: tissue IHC). |
| Brown haze covers several tissue compartments or obscures the boundary between nuclei and surrounding tissue. | Diffuse background prevents a confident nuclear localisation call. Review the detection-only control and staining development, then reassess individual nuclei on the same section (general IHC practice). HPA's supported reliability describes agreement between antibody staining and RNA expression data; it does not validate an uninterpretable slide (HPA: tissue IHC reliability). |
| No nuclear signal is visible in a suitable section containing an expected high-staining population. | Consider a technical failure before concluding that CBX3 is absent: HPA reports high staining in alveolar type I cells, cytotrophoblasts, melanocytes, and pachytene spermatocytes (HPA: tissue IHC). Confirm the relevant cells are present, then review the IHC workflow and controls (general IHC practice). One failed section cannot overturn the reported tissue pattern. |
💡Expected CBX3 appearanceCall a positive result when chromogen is predominantly nuclear in identifiable cells, with clear signal in an HPA high-staining population such as placental cytotrophoblasts; a dominant nonnuclear deposit or indiscriminate haze is suspect (HPA: tissue IHC; UniProt Q13185: nucleus; general IHC practice).
| Tissue and cell selection (HPA: tissue IHC) | Choose an identifiable high-staining population for the positive reference: lung alveolar type I cells, placental cytotrophoblasts, skin melanocytes, or testicular pachytene spermatocytes (HPA: tissue IHC). Hepatocytes and skeletal myocytes are reported low, not negative; weak staining there does not establish assay failure (HPA: tissue IHC). |
| Compartment and chromatin context (UniProt Q13185; HPA: subcellular ICC-IF) | Score nuclei rather than a membrane or secreted compartment: UniProt places CBX3 in the nucleus and reports no transmembrane segment. UniProt says it associates with euchromatin and is largely excluded from constitutive heterochromatin; HPA ICC-IF supports mainly nucleoplasmic localisation with additional nuclear bodies. Nuclear bodies are an IF observation, not a required chromogenic IHC feature. |
| Antibody evidence (HPA: antibody validation) | HPA lists IHC support for both HPA004902 and CAB001973, and ICC support for HPA004902; no ICC status is supplied for CAB001973 (HPA: antibody validation). This supports the listed applications but does not establish identical staining with every antibody or guarantee performance under a different IHC workflow (general IHC practice). |
| Protein annotation limits (UniProt Q13185) | UniProt reports no isoforms, signal peptide, propeptide, or glycosylation sites, and identifies a chain spanning residues 2–183 (UniProt Q13185: processing and isoforms). These annotations provide no evidence for a shed staining compartment or an isoform-specific tissue pattern. The supplied record does not locate the antibody epitope or establish CBX3-specific fixation or retrieval sensitivity. |
| IF/ICC Q: What localisation should a separate IF/ICC guide assess? (HPA: subcellular ICC-IF) | A: Mainly nucleoplasmic signal, with additional nuclear bodies, is supported by HPA ICC-IF images from A-431, U-251MG, and U2OS cells (HPA: subcellular ICC-IF). This is a localisation comparison for the separate IF/ICC application; it does not supply an IF protocol or change the nuclear IHC scoring criterion. |
| Situation | Likely cause | Next action |
|---|---|---|
| Expected positive cells lack nuclear chromogen. | The high-staining population may be absent from the section, or an IHC step may have failed (HPA: tissue IHC; general IHC practice). | Identify the cells on a counterstained section, then check the positive control, antibody preparation, retrieval record, and detection steps (general IHC practice). No CBX3-specific retrieval condition or fixation effect is supplied. |
| Signal is mainly outside nuclei. | The observed compartment disagrees with the nuclear pattern; nonspecific staining or a detection artifact is possible (UniProt Q13185: nucleus; HPA: tissue IHC; general IHC practice). | Recheck cell boundaries and the detection-only control, then assess whether nuclear staining is independently visible (general IHC practice). Do not score the nonnuclear deposit as CBX3 solely because it is intense. |
| Brown deposit appears when primary antibody is omitted. | Endogenous activity or another detection-system contribution is possible (general IHC practice). | Address the source indicated by the detection-only control, then repeat the comparison with the same tissue type (general IHC practice). HPA tissue staining levels do not identify the source of that deposit. |
| Diffuse background makes nuclei hard to distinguish. | Excess background or overdevelopment may obscure the expected nuclear pattern (general IHC practice; UniProt Q13185: nucleus). | Review blocking, washing, antibody dilution, and chromogen development under the chosen IHC workflow; compare controls before assigning nuclear scores (general IHC practice). The supplied evidence specifies no dilution or development time. |
| A low-staining tissue appears negative or much brighter than expected. | Low is an HPA staining category, not a formal negative control; technical background or cell identification may affect the comparison (HPA: tissue IHC; general IHC practice). | Compare identified cells with an HPA high-staining population on a suitable control section and review background controls (HPA: tissue IHC; general IHC practice). Avoid calling the tissue CBX3-negative or the brighter result specific from intensity alone. |
| Fine nuclear bodies seen by IF/ICC cannot be resolved in chromogenic IHC. | HPA reports nuclear bodies as an additional ICC-IF location, while its tissue IHC profile describes ubiquitous nuclear expression (HPA: subcellular ICC-IF; HPA: tissue IHC). | Score the predominant nuclear IHC pattern in identifiable cells; reserve assessment of nuclear bodies for the separate IF/ICC guide (HPA: tissue IHC; HPA: subcellular ICC-IF). |
Experiment evidence
IHC & IF images showing CBX3 staining
A01142 · IHCIHC analysis of HP1 gamma using anti-HP1 gamma antibody (A01142). HP1 gamma was detected in paraffin-embedded section of human mammary cance
A01142 · IHCIHC analysis of HP1 gamma using anti-HP1 gamma antibody (A01142). HP1 gamma was detected in paraffin-embedded section of human rectal cancer
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.
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 placenta first and assess cytotrophoblast nuclei, which should stain (HPA: High in cytotrophoblasts). HPA detects CBX3 in all 44 scored tissues, so there is no documented negative tissue; use no-primary and isotype controls, and treat cells without nuclear staining on the positive slide as internal background checks rather than established CBX3-negative cells (HPA: no negative rows; UniProt Q13185: nucleus).
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: Run a secondary-only slide and a rabbit immunoglobulin isotype control matched to the primary’s clonality, plus a CBX3 knockout sample if available or a validated peptide-block control (A01142 caption: rabbit primary; standard IHC controls). For the caption’s SABC/DAB detection, block endogenous peroxidase and check for endogenous biotin background in placenta (A01142 caption: SABC/DAB; standard IHC practice).
⚠️Feasibility: The tissue-IHC caption provides a starting retrieval condition of citrate buffer at pH 6 for 20 minutes, but does not establish that retrieval is required for every specimen (A01142 caption). Its fixative is unreported, and the supplied evidence reports no CBX3-specific fixation window or fixation effect (A01142 caption). Whether frozen sections or IF are easier is unreported; placenta may show endogenous biotin background with the caption’s SABC detection, while HPA ICC-IF supports predominantly nucleoplasmic staining with additional nuclear bodies (standard IHC practice; A01142 caption; HPA subcellular).
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
Troubleshoot CBX3 staining in paraffin sections by checking nuclear localisation, the documented retrieval conditions, and controls before comparing chromogenic signal across samples.
Which retrieval conditions should I try first for weak CBX3 staining?
Start with heat mediated retrieval in citrate buffer at pH 6 for 20 minutes (datasheet A01142). The documented paraffin section image used these conditions before incubation with 1 μg/ml catalog antibody overnight at 4°C (datasheet A01142). If nuclear staining remains weak, compare a longer retrieval or an alternative buffer on adjacent sections while keeping detection and exposure to chromogen constant (standard IHC practice). Include a section with an expected nuclear signal, such as placenta cytotrophoblasts, and assess whether any gain in signal also raises background (HPA: High in cytotrophoblasts; standard IHC practice).
Could fixation explain weak or uneven CBX3 staining in my paraffin sections?
Target specific fixation sensitivity is unknown because the selected paraffin section caption does not state a fixative (datasheet A01142). Record the fixative, fixation duration, tissue thickness, and processing history for each sample before comparing nuclear signal (standard IHC practice). Compare sections processed together under the documented citrate pH 6, 20 minute retrieval condition, then inspect morphology and nuclear staining side by side (datasheet A01142; standard IHC practice). If fixation histories differ, treat a weaker result as inconclusive until matched processing or an independent control resolves the difference; neither nuclear localisation nor protein modifications establish a fixation effect (UniProt Q13185 localisation and modified residues; standard IHC practice).
Where should CBX3 appear, and how should I investigate cytoplasmic staining?
Expect predominantly nuclear chromogenic staining: CBX3 is nuclear and associates with euchromatin, while tissue IHC shows ubiquitous nuclear expression (UniProt Q13185 localisation; HPA: ubiquitous nuclear expression). The supported subcellular pattern is mainly nucleoplasmic with additional nuclear bodies, so a diffuse or punctate nuclear pattern can merit evaluation (HPA subcellular: supported locations). Compare the brown signal with the nuclear counterstain and inspect several intact fields before scoring (standard IHC practice). If cytoplasmic or extracellular colour dominates, review the no primary control, tissue pigment, and chromogen development before calling it CBX3; a mitotic pole association is only potential evidence (UniProt Q13185 localisation; standard IHC practice).
How do I troubleshoot epitope masking without assuming a CBX3 isoform difference?
The supplied record lists 0 isoforms and does not map this antibody's epitope, so do not assign staining differences to an isoform or a particular residue (UniProt Q13185 isoforms; datasheet A01142). CBX3 contains a chromo domain at residues 30–88 and a chromoshadow domain at 121–179, with annotated modified residues, but their effect on this antibody's tissue staining is unknown (UniProt Q13185 domains and modified residues). Compare retrieval conditions on serial sections, beginning with citrate pH 6 for 20 minutes, and keep antibody concentration fixed (datasheet A01142; standard IHC practice). Interpret changed staining alongside nuclear localisation and background controls rather than inferring which epitope was exposed (HPA: ubiquitous nuclear expression; standard IHC practice).
How should I adapt CBX3 localisation checks to multiplex IF?
For the separate IF/ICC workflow, pair CBX3 with a validated marker of the expected cell type, such as a cytotrophoblast marker when examining placenta, and score overlap within individual nuclei (HPA: High in cytotrophoblasts; HPA subcellular: nucleoplasm). Choose fluorophores after checking tissue autofluorescence and include single label controls to evaluate spectral bleed through (standard IF practice). CBX3 has no transmembrane segment and is mainly nucleoplasmic, so permeabilise sufficiently for antibody access to nuclear epitopes while checking that nuclear morphology remains intact (UniProt Q13185 topology; HPA subcellular: nucleoplasm; standard IF practice). Optimise IF fixation and permeabilisation independently; the paraffin section caption supplies no IF fixation condition (datasheet A01142).
What should I check when DAB background obscures CBX3 nuclei?
The documented chromogenic image used 10% goat serum blocking, 1 μg/ml primary antibody, a biotinylated secondary, and DAB development (datasheet A01142). Run a no primary control, check endogenous peroxidase blocking, and compare signal before and after chromogen development to identify nonspecific colour (standard IHC practice). With a biotin based detection system, assess whether endogenous biotin contributes to staining and use appropriate blocking or a different detection chemistry if necessary (datasheet A01142 detection method; standard IHC practice). Judge any adjustment against nuclear signal in intact cells, because the supported tissue pattern is nuclear rather than extracellular (HPA: ubiquitous nuclear expression; standard IHC practice).
How can I quantify CBX3 IHC across samples with different cellularity? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and scoring region before reading the slides, then record nuclear intensity and the percentage of positive nuclei for an H-score (standard IHC practice; HPA: ubiquitous nuclear expression). If spatial abundance matters, report positive nuclei per mm² alongside the total nuclei counted in that region (standard IHC practice). Normalise comparisons to the number of eligible intact cells or measured tissue area, and keep staining and image analysis settings consistent across samples (standard IHC practice). Report cell type separately where possible, since HPA describes High staining in placenta cytotrophoblasts but Low staining in liver hepatocytes (HPA: cytotrophoblasts High; hepatocytes Low).
When is an apparent CBX3 positive cell likely to be an artefact?
A convincing positive has chromogen over an intact nucleus within an identifiable cell, consistent with the reported ubiquitous nuclear tissue pattern and mainly nucleoplasmic location (HPA: ubiquitous nuclear expression; HPA subcellular: nucleoplasm). Treat isolated cytoplasmic colour, stained debris, and signal confined to section edges or necrotic areas as suspect until controls and morphology support the call (standard IHC practice). Check a no primary section and the peroxidase block when brown signal follows tissue pigment or endogenous enzyme activity (standard IHC practice). Compare plausible nuclear staining across cell types without demanding uniform intensity, because HPA reports both High and Low populations and rates tissue IHC reliability as Supported (HPA: tissue profile and reliability).
Boster reagents
Best CBX3 / Chromobox protein homolog 3 IHC Antibodies
A01142 has pictured IHC results in human, mouse and rat paraffin sections, plus an IF result in U20S cells (A01142 image captions).
Real IHC data
Anti-HP1 gamma/CBX3 Antibody ®
Cat # A01142
The rendered SKU is A01142; its IHC captions show human mammary and rectal cancer and mouse and rat small intestine in paraffin sections (A01142 IHC captions). Its IF caption shows U20S cells, and the catalog lists Human, Mouse and Rat reactivity (A01142 IF caption; catalog reactivity).
Which to pick: Choose A01142 for tissue IHC and pictured cross-species examples: its captions document paraffin sections from Human, Mouse and Rat, but do not report the fixative (A01142 IHC captions). Choose A01142 for IF/ICC because its application list includes both and its IF caption shows U20S cells (A01142 catalog applications; IF caption). M01142-1 is a rabbit monoclonal listed for IHC and Human, Mouse and Rat reactivity, but has no supplied IHC image caption (M01142-1 catalog).
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.
Immunohistochemistry protocol sheet for CBX3 (Chromobox protein homolog 3). Expected localisation Nuclei across tissues (HPA tissue IHC). Antigen retrieval: Citrate pH 6 HIER, heat-mediated (datasheet A01142). Validated IHC antibody: A01142 at 0.5-1μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Lung (Alveolar cells type I); negative None in HPA (detected in all 44 tissues); use no-primary + isotype controls. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human mammary cancer tissue; fixative not specified (datasheet A01142); 2. Fixation: Image fixative and duration unreported (datasheet A01142); 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: Citrate pH 6 , 20 min (datasheet A01142); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A01142); 8. Primary antibody: Rabbit anti-CBX3, 0.5-1μg/ml (datasheet A01142); 9. Primary incubation: Overnight at 4 °C (datasheet A01142). Watch for: Fixation: Keep fixation conditions consistent across sections. (standard IHC practice; not target-specific); Caveat: Ubiquitous nuclear signal limits unstained-cell controls (HPA tissue IHC); Isoform / epitope: No isoforms annotated; chain spans residues 2–183 (UniProt); Regulation: No expression regulator annotated (UniProt). Evidence: UniProt Q13185, HPA tissue IHC · Supported, HPA ICC-IF · Mainly localized to the nucleoplasm. In addition localized to the…, Datasheet A01142, PMC6540543, PMC5920882. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/CBX3/immunohistochemistry-ihc-guide.