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CCAR2 / Cell cycle and apoptosis regulator protein 2 · IHC design guide
Design Immunohistochemistry for CCAR2
Plan CCAR2 IHC in paraffin sections around predominantly nuclear staining (HPA tissue IHC), using 1–2 μg/ml of the catalog antibody as a starting range (datasheet A03412-1). Respiratory epithelial cells show high staining, while alveolar cells have no detected staining (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
CCAR2 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 tissue staining (HPA tissue IHC) | |
| Staining pattern | Broad nuclear staining across tissue cell types (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A03412-1) | |
| Positive control | Bronchus+4 more · see all | |
| Negative control | Lung |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent across sections (standard IHC practice; not target-specific) | |
| Caveat | Alveolar cells may lack staining despite the broad tissue profile (HPA tissue IHC) | |
| Regulation | Expression rises with gastric carcinoma progression (UniProt) | |
| Isoform / epitope | 2 isoforms; epitope coverage is unspecified (UniProt) |
Section 1
Recommended CCAR2 IHC & IF Protocols
The catalog antibody has an IHC-P protocol (datasheet A03412-1); one published CCAR2 IHC protocol uses paraffin-embedded breast tissue (PMC13229996).
| Sample | Paraffin-embedded human gall bladder adenosquamous carcinoma tissue; fixative not specified (datasheet A03412-1) |
| Fixation | Image fixative and duration unreported (datasheet A03412-1); 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 A03412-1); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A03412-1) |
| Primary antibody | Rabbit anti-CCAR2, 1-2 μg/ml (datasheet A03412-1) |
| Primary incubation | Overnight at 4 °C (datasheet A03412-1) |
| Detection | Streptavidin-biotin complex (SABC), DAB chromogen (datasheet A03412-1) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | CCAR2-positive staining in respiratory epithelial cells of bronchus (HPA tissue IHC: High). HPA tissue profile: Ubiquitous nuclear expression. No signal in the no-primary control. |
💡Decision noteStart with EDTA pH 8.0 heat retrieval for the catalog antibody (datasheet A03412-1); use citrate pressure retrieval when reproducing the published protocol (PMC13229996).
Section 2
What Is the Expected CCAR2 Staining Pattern?
CCAR2 should appear predominantly in nuclei across many normal tissues, with strong staining in selected epithelial, glandular and neuronal cells (HPA tissue IHC: ubiquitous nuclear expression; High in listed cell types). The tissue profile has Enhanced reliability, reflecting agreement between antibody staining and RNA expression (HPA tissue IHC). CCAR2 has no transmembrane segment; UniProt also lists cytoplasmic localisation under specific conditions (UniProt Q8N163 topology and subcellular location).
| Clear nuclear staining in bronchial respiratory epithelium or esophageal squamous epithelium. | This matches the expected compartment and cells; both are reported High, though intensity can vary across a section (HPA tissue IHC; standard IHC practice). |
| Predominantly cytoplasmic staining in an untreated section, with little nuclear signal. | This differs from the HPA tissue profile. Check staining specificity and slide quality; cytoplasmic CCAR2 is possible in defined contexts, including UV-induced apoptosis (HPA tissue IHC; UniProt Q8N163 subcellular location). |
| Strong staining chiefly in lung alveolar cells while adjacent expected-positive cells are unstained. | HPA reports alveolar cells as Not detected. Consider nonspecific binding or endogenous detection activity, then compare the compartment and appropriate controls; Not detected is not proof of absolute absence (HPA tissue IHC; standard IHC practice). |
| Diffuse colour over nuclei, cytoplasm and tissue spaces without clear cell boundaries. | This is difficult to score as CCAR2. In chromogenic IHC, inadequate washing, nonspecific reagent binding or endogenous detection activity can produce background (standard IHC practice). |
| No nuclear signal in bronchial respiratory epithelial cells despite an interpretable section. | Those cells are reported High, so a blank result warrants checking the assay before concluding biological absence. Review controls, retrieval, detection and reagent handling (HPA tissue IHC; standard IHC practice). |
💡Expected CCAR2 appearanceCall a result positive when distinct nuclear staining is visible in expected cells, especially High bronchial respiratory epithelial or esophageal squamous epithelial cells; diffuse colour without a nuclear pattern is suspect (HPA tissue IHC; standard IHC practice).
| Tissue and cell selection | High staining is reported in bronchial respiratory epithelium, several glandular and neuronal populations; alveolar cells are Not detected, while some other cells are Low (HPA tissue IHC). Select and interpret controls at the cell-type level. |
| Subcellular context | The reference tissue pattern is nuclear (HPA tissue IHC). UniProt reports recruitment to chromatin after UV, cytoplasmic sequestration with MCC and cytoplasmic translocation during UV-induced apoptosis (UniProt Q8N163 subcellular location); those contexts should be documented before interpreting a shift. |
| IF/ICC Q&A: what pattern is expected? | Nucleoplasm and nuclear bodies are the enhanced locations in cultured-cell imaging (HPA subcellular ICC-IF). This is an IF/ICC localisation reference, not evidence that nuclear bodies must resolve in chromogenic tissue IHC (HPA subcellular ICC-IF; HPA tissue IHC). |
| Antibody validation | HPA019907 and HPA019943 have Enhanced IHC status; CAB072827 has Supported IHC status (HPA antibodies). These ratings support the reported pattern but do not establish identical performance for another antibody or preparation (HPA antibodies; standard IHC practice). |
| Isoforms and epitope coverage | UniProt lists two CCAR2 isoforms and one annotated chain spanning residues 1–923 (UniProt Q8N163 isoforms and processing). The supplied record does not locate this assay's epitope, so isoform coverage and any effect on staining cannot be assigned. |
| Situation | Likely cause | Next action |
|---|---|---|
| No signal in an expected-positive bronchus control. | Assay failure is plausible because respiratory epithelial cells are reported High; the observation alone cannot identify the failed step (HPA tissue IHC). | Confirm the positive-control section and run details, then review retrieval, antibody application and chromogenic detection one step at a time (standard IHC practice). |
| Only cytoplasmic staining is visible. | This conflicts with the usual nuclear tissue profile, although UniProt describes cytoplasmic localisation in specific biological contexts (HPA tissue IHC; UniProt Q8N163 subcellular location). | Check nuclear counterstain and cell boundaries, compare an expected-positive control, and record relevant UV, apoptosis or MCC context before assigning the signal to CCAR2 (standard IHC practice; UniProt Q8N163 subcellular location). |
| Lung alveolar cells appear strongly positive. | HPA records alveolar cells as Not detected; nonspecific binding or endogenous chromogenic activity is possible (HPA tissue IHC; standard IHC practice). | Inspect the nuclear pattern and use appropriate detection and antibody controls; interpret this as a discrepancy requiring validation, not an automatic biological negative or positive (standard IHC practice; HPA tissue IHC). |
| Broad, diffuse chromogen obscures nuclear detail. | Excess background can arise from nonspecific binding, incomplete washing or endogenous detection activity (standard IHC practice). | Review blocking, washing and detection controls; score CCAR2 only where nuclei can be distinguished from background (standard IHC practice; HPA tissue IHC). |
| Weak staining in pancreatic exocrine glandular cells. | Low staining is reported for this cell type, so weak signal alone need not indicate an assay failure (HPA tissue IHC). | Check a High cell-type control before adjusting the assay; interpret the pancreatic result against its own reported level (HPA tissue IHC; standard IHC practice). |
| Two antibodies give different nuclear patterns. | Antibody performance can differ; HPA validation status applies to the listed antibodies and does not resolve every section-level discrepancy (HPA antibodies; standard IHC practice). | Compare the same tissue and cell types, review controls and antibody-specific instructions, then report unresolved disagreement rather than merging the scores (standard IHC practice). |
Experiment evidence
IHC & IF images showing CCAR2 staining
A03412-1 · IHCIHC analysis of DBC-1/CCAR2 using anti-DBC-1/CCAR2 antibody (A03412-1). DBC-1/CCAR2 was detected in a paraffin-embedded section of human gal
A03412-1 · IHCIHC analysis of DBC-1/CCAR2 using anti-DBC-1/CCAR2 antibody (A03412-1). DBC-1/CCAR2 was detected in a paraffin-embedded section of human liv
A03412-1 · IFIF analysis of DBC-1/CCAR2 using anti-DBC-1/CCAR2 antibody (A03412-1). DBC-1/CCAR2 was detected in an immunocytochemical section of U20S cel
A30523 · IHCImmunohistochemical analysis of paraffin-embedded human liver cancer. 1, Antibody was diluted at 1:200(4° overnight). 2, Tris-EDTA,pH9.0 was
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000158941-CCAR2/tissue/cerebral+cortex#imid_5424500
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000158941-CCAR2/tissue/colon#imid_5424426
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000158941-CCAR2/tissue/liver#imid_5423859
HPA · PancreasHuman Protein Atlas IHC, Pancreas. Source: https://www.proteinatlas.org/ENSG00000158941-CCAR2/tissue/pancreas#imid_5424386
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA019907; green = CCAR2, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA019907; green = CCAR2, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for CCAR2 IHC & IF
🧪Run bronchus first: respiratory epithelial cells should stain (HPA: High in bronchus respiratory epithelial cells). Use lung alveolar cells as the biological comparison (HPA: Not detected); on the bronchus slide, internal negative cells should lack DAB staining, but the supplied HPA rows do not identify a specific negative cell type there (HPA: bronchus row).
Positive control tissue: Bronchus (Respiratory epithelial cells, HPA High)
Negative control tissue: Lung (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show CCAR2 in A-431, U-251MG, U2OS, HeLa, MCF-7, U2OS, siRNA 1 (10x), U2OS, siRNA 2 (10x), U2OS, scrambled (10x), with annotated localisation: Nucleoplasm (enhanced), Nuclear bodies (enhanced) (HPA subcellular).
Technical controls: Include no-primary (secondary-only) and concentration-matched rabbit IgG isotype controls, plus CCAR2 knockout tissue or a validated peptide-block control where available (selected-SKU caption: rabbit primary antibody). For the biotin-based DAB workflow, check endogenous peroxidase and biotin background in bronchus (selected-SKU caption: biotinylated secondary, streptavidin–biotin detection and DAB).
⚠️Feasibility: A CCAR2-specific fixation window or fixation effect is unreported, and the selected-SKU paraffin-section caption does not state the fixative (selected-SKU caption: fixative not stated). The reported IHC conditions use heat-mediated EDTA retrieval at pH 8.0; whether retrieval is required for every specimen is unreported (selected-SKU caption: EDTA retrieval). Frozen sections are not established as easier by the supplied evidence; IF can assess the reported nucleoplasm and nuclear-body localization, while bronchus requires attention to endogenous detection background (HPA subcellular: nucleoplasm and nuclear bodies; selected-SKU caption: biotin-based DAB detection).
HPA tissue IHC evidence for CCAR2
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 |
|---|---|---|---|---|
| Bronchus | Respiratory epithelial cells | High | Protein (IHC) | HPA → |
| Cerebellum | Cells in molecular layer | High | Protein (IHC) | HPA → |
| Cerebral cortex | Neuronal cells | High | Protein (IHC) | HPA → |
| Epididymis | Glandular cells | High | Protein (IHC) | HPA → |
| Esophagus | Squamous epithelial cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Lung | Alveolar cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced CCAR2 IHC Tips
Use the paraffin-section protocol as the IHC starting point, then assess CCAR2 staining by cell type, compartment and matched controls.
What retrieval should I try first if nuclear CCAR2 staining is weak?
Start with heat-mediated EDTA at pH 8.0 for paraffin sections (datasheet A03412-1). The selected tissue image used this retrieval before overnight incubation at 4°C with 2 μg/ml catalog antibody (datasheet A03412-1). Keep retrieval and detection conditions consistent across comparison sections, then check whether expected nuclear staining appears in glandular cells of gallbladder or respiratory epithelial cells of bronchus (HPA: High in both cell types; standard IHC practice). If staining remains weak, compare a cautious citrate-buffer retrieval trial on matched sections, judging nuclear signal alongside tissue morphology and background (standard IHC practice).
How should I handle uncertain fixation when CCAR2 staining varies between sections?
The selected paraffin-section caption does not report a fixative, so target-specific fixation sensitivity is unknown (datasheet A03412-1). Record the available fixation and processing history for each specimen, and compare sections processed together before attributing a signal difference to CCAR2 biology (standard IHC practice). Apply the documented EDTA pH 8.0 retrieval consistently, then inspect nuclear staining and tissue preservation on adjacent sections (datasheet A03412-1; HPA: ubiquitous nuclear expression; standard IHC practice). Neither the HPA tissue pattern nor CCAR2 topology and modifications establish how a particular fixative affects this antibody (HPA: tissue IHC; UniProt Q8N163 topology and modified residues).
Which staining compartment should count as a CCAR2 positive result?
Score nuclear staining first: tissue IHC shows ubiquitous nuclear expression, while cell imaging places CCAR2 in the nucleoplasm and nuclear bodies (HPA: tissue IHC and subcellular localisation). Use the nuclear counterstain to separate nuclear signal from adjacent cytoplasm, and record the fraction of cells with convincing nuclear staining (standard IHC practice). Cytoplasmic staining deserves separate assessment because CCAR2 can be sequestered there with MCC or translocate there during UV-induced apoptosis (UniProt Q8N163 subcellular annotation). Do not interpret diffuse cytoplasmic color alone as equivalent to the expected nuclear pattern; check morphology, cell type and matched controls first (HPA: tissue IHC; standard IHC practice).
Could isoforms or epitope changes explain discordant CCAR2 staining?
CCAR2 has 2 annotated isoforms, but the supplied antibody evidence does not map its epitope or establish isoform recognition (UniProt Q8N163 isoforms; datasheet A03412-1). It also lists modified residues, including acetyllysine at position 112 and phosphoserine at position 124; their effect on this antibody is unreported (UniProt Q8N163 modified residues; datasheet A03412-1). When samples disagree, compare matched sections under the same retrieval and detection conditions before proposing epitope masking (standard IHC practice). Ask for documented epitope mapping or use an independently validated antibody against a distinct region before assigning an isoform-specific or modification-specific staining pattern (standard IHC practice).
How can I check the IHC pattern using multiplex IF?
For an IF comparison, pair CCAR2 with a validated epithelial cell marker in bronchus or gallbladder and use a nuclear counterstain to assign signal to individual cells (HPA: High in respiratory and gallbladder glandular cells; standard IF practice). Choose a far-red fluorophore when tissue autofluorescence compromises shorter-wavelength channels, and include single-stain controls to assess channel bleed-through (standard IF practice). CCAR2 has no transmembrane segment and is primarily nuclear, so permeabilise for access to its intracellular epitope while checking that nuclear morphology remains intact (UniProt Q8N163 topology; HPA: subcellular localisation; standard IF practice). Compare the resulting nuclear pattern with the paraffin-section IHC pattern without assuming identical processing requirements across methods (HPA: tissue IHC; standard IF practice).
What should I check when DAB appears outside CCAR2 positive nuclei?
Compare suspect color with the expected nuclear pattern in tissue sections and with the nucleoplasmic and nuclear-body pattern reported by cell imaging (HPA: tissue IHC and subcellular localisation). The selected image used 10% goat serum blocking, a biotinylated secondary, a streptavidin-biotin complex and DAB, so check matched controls for secondary-system background (datasheet A03412-1; standard IHC practice). Include a peroxidase block and a primary-antibody omission control when evaluating DAB deposits; these are general chromogenic IHC checks (standard IHC practice). Inspect folds, section edges and damaged areas separately, and judge whether any apparent cytoplasmic signal follows intact cells rather than nonspecific deposits (standard IHC practice).
How should I quantify CCAR2 across samples with different cell composition? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring; CCAR2 tissue staining is broadly nuclear, but reported levels differ by cell type (HPA: tissue IHC). For chromogenic sections, report the percentage of positive nuclei and an H-score combining nuclear staining intensity with the percentage at each intensity, using a 0–300 scale (standard IHC practice). Normalise counts to the number of eligible cells in the defined compartment, or report positive-cell density per mm² when area is the denominator (standard IHC practice). Keep thresholds, counterstain assessment and exclusion of damaged regions consistent across sections, and report cytoplasmic staining separately if it is studied (standard IHC practice; UniProt Q8N163 subcellular annotation).
How do I distinguish true CCAR2 staining from an artefact?
A credible result favors intact nuclei in an appropriate cell population: HPA reports high staining in gallbladder glandular cells and no detected staining in lung alveolar cells (HPA: tissue IHC). Check the nuclear counterstain and tissue architecture before calling a brown deposit cellular, especially at section edges or within necrotic regions (standard IHC practice). Compare a primary-antibody omission control and the peroxidase-blocked section when deposits suggest endogenous enzyme activity or detection-system background (standard IHC practice). Cytoplasmic signal is biologically possible under specified conditions, including MCC association or UV-induced apoptosis, but requires separate evidence before interpretation in an unstated specimen context (UniProt Q8N163 subcellular annotation).
Boster reagents
Best CCAR2 / Cell cycle and apoptosis regulator protein 2 IHC Antibodies
Anti-CCAR2 antibodies have paraffin-section IHC images from human cancers and mouse brain (A03412-1 and A30523 IHC captions); A03412-1 also has cell-based IF data (A03412-1 IF caption).
Real IHC data
Anti-DBC-1/CCAR2 Antibody ®
Cat # A03412-1
Real IHC data
Anti-KIAA1967 CCAR2 Antibody
Cat # A30523
A03412-1 has IHC images from human gall bladder adenosquamous carcinoma, liver cancer and lung cancer, and mouse brain, plus an IF image from U20S cells (A03412-1 image captions). A30523 has an IHC image from paraffin-embedded human liver cancer (A30523 IHC caption).
Which to pick: For tissue IHC, choose A03412-1 for the broader set of pictured samples or A30523 for the pictured human liver cancer sample; both captions describe paraffin sections, and neither reports the fixative (A03412-1 and A30523 IHC captions). For IF/ICC, A03412-1 has a cell-based IF image, while A30523 lists IF and ICC without an IF image (A03412-1 IF caption; A30523 applications and image payload). For work across species, A03412-1 lists Human, Mouse, Rat and Monkey reactivity, though its stated IHC dilution covers Human, Mouse and Rat; A30523 lists Human, Mouse and Rat reactivity and is described as polyclonal (A03412-1 dilution and reactivity data; A30523 reactivity and dilution data).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q8N163 (CCAR2_HUMAN, Cell cycle and apoptosis regulator protein 2).
- Human Protein Atlas. CCAR2 tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. CCAR2 subcellular location (ICC-IF): Localized to the nucleoplasm and nuclear bodies..
- Human Protein Atlas. CCAR2 antibody validation summary (4 antibodies).
- Sirtuin 1 stimulates the proliferation and the expression of glycolysis genes in pancreatic neoplastic lesions. Oncotarget 2016 — PMC5342700.
- An anoikis-related gene signature predicts prognosis and immunotherapy response, and identifies CCAR2 as a therapeutic target in triple-negative breast cancer. Frontiers in immunology 2026 — PMC13229996.
- The expression of DBC1/CCAR2 is associated with poor prognosis of ovarian carcinoma. Journal of ovarian research 2015 — PMC4335761.
- CCAR2 negatively regulates IL-8 production in cervical cancer cells. Oncotarget 2018 — PMC5787426.
- PubMed PMID:14702039 — UniProt-cited evidence.
- PubMed PMID:17974005 — UniProt-cited evidence.
- PubMed PMID:16421571 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for CCAR2 (Cell cycle and apoptosis regulator protein 2). Expected localisation Predominantly nuclear tissue staining (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A03412-1). Validated IHC antibody: A03412-1 at 1-2 μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Bronchus (Respiratory epithelial cells); negative Lung. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human gall bladder adenosquamous carcinoma tissue; fixative not specified…; 2. Fixation: Image fixative and duration unreported (datasheet A03412-1); 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 A03412-1); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A03412-1); 8. Primary antibody: Rabbit anti-CCAR2, 1-2 μg/ml (datasheet A03412-1); 9. Primary incubation: Overnight at 4 °C (datasheet A03412-1). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific); Caveat: Alveolar cells may lack staining despite the broad tissue profile (HPA tissue IHC); Isoform / epitope: 2 isoforms; epitope coverage is unspecified (UniProt); Regulation: Expression rises with gastric carcinoma progression (UniProt). Evidence: UniProt Q8N163, HPA tissue IHC · Enhanced, HPA ICC-IF · nucleoplasm and nuclear bodies., Datasheet A03412-1, PMC13229996. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/CCAR2/immunohistochemistry-ihc-guide.