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CACYBP / Calcyclin-binding protein · IHC design guide
Design Immunohistochemistry for CACYBP
Plan paraffin-section CACYBP staining around the mainly cytoplasmic tissue pattern and differences between cell types (HPA tissue IHC). The guide covers antibody dilution, detection and scoring using the 0.5–1 μg/mL IHC range for RP1104 (datasheet RP1104).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
CACYBP 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 | Tissue: mainly cytoplasmic (HPA tissue IHC); nuclear localisation is possible (UniProt) | |
| Staining pattern | Cytoplasmic in most tissues; high in endocrine and neuronal cells (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet RP1104) | |
| Positive control | Appendix+4 more · see all | |
| Negative control | Heart muscle+3 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent across sections. (standard IHC practice; not target-specific) | |
| Caveat | Cell-type mix can mask high endocrine or neuronal staining (HPA tissue IHC) | |
| Regulation | Expression regulation unannotated (UniProt) | |
| Isoform / epitope | 3 isoforms; epitope coverage is unspecified (UniProt) |
Section 1
Recommended CACYBP IHC & IF Protocols
The catalog antibody protocol uses EDTA pH 8.0 retrieval (datasheet: RP1104). Four published CACYBP IHC protocols below use citrate pH 6.0 retrieval (PMC10779320; PMC7051089; PMC3996359; PMC10299707).
| Sample | Paraffin-embedded human intestinal cancer tissue; fixative not specified (datasheet RP1104) |
| Fixation | Image fixative and duration unreported (datasheet RP1104); 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 RP1104); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet RP1104) |
| Primary antibody | Rabbit anti-CACYBP, 0.5-1μg/ml (datasheet RP1104) |
| Primary incubation | Overnight at 4 °C (datasheet RP1104) |
| Detection | Streptavidin-biotin complex (SABC), DAB chromogen (datasheet RP1104) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | CACYBP-positive staining in endocrine cells of appendix (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression in most tissues. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA pH 8.0 for the catalog antibody (datasheet: RP1104); evaluate citrate pH 6.0 when adapting a published antibody protocol (PMC10779320; PMC7051089; PMC3996359; PMC10299707).
Section 2
What Is the Expected CACYBP Staining Pattern?
CACYBP is mainly cytoplasmic in tissue IHC (HPA: tissue profile) and can also localize to the nucleus (UniProt Q9HB71: subcellular location). Expect staining in selected endocrine, neuronal, squamous epithelial, and germinal center cells (HPA: High). HPA rates the tissue profile Enhanced, with medium consistency between antibody staining and RNA expression (HPA: tissue reliability). CACYBP has no transmembrane segment (UniProt Q9HB71: topology).
| Cytoplasmic staining in colon endocrine cells, cortical neurons, or lymph node germinal center cells. | This fits reported high expression in those cell populations (HPA: tissue IHC). Assess the named cells rather than assigning one intensity to the entire section; neighboring cell types need not share their staining level (HPA: cell-specific tissue IHC). |
| Signal is confined to cell borders, or only nuclei stain across the section. | A dominant border pattern conflicts with cytoplasmic tissue staining and the absence of a transmembrane segment (HPA: tissue profile; UniProt Q9HB71: topology). Nuclear signal can be plausible (UniProt Q9HB71: subcellular location), but nuclear-only staining should be checked against a known-positive cytoplasmic pattern before interpretation. |
| Strong staining appears in cardiomyocytes or skeletal myocytes while expected positive cells are faint. | HPA reports these muscle cells as not detected (HPA: heart and skeletal muscle IHC). Consider antibody cross-reactivity or endogenous chromogenic activity (general IHC practice), while allowing that HPA observations are reference patterns, not absolute exclusions for every specimen. |
| Color is spread across tissue and empty spaces without recognizable cellular localization. | Diffuse deposit cannot establish CACYBP localization; nonspecific binding, incomplete blocking, or detection background are possibilities (general IHC practice). Compare with a reagent control and inspect whether the deposit follows the expected cytoplasm of positive cells (HPA: tissue profile). |
| No staining is visible in a nominated positive region, such as esophageal squamous epithelium. | Esophageal squamous epithelial cells are reported High (HPA: esophagus IHC), so absence there makes this run difficult to interpret. Check tissue identity, antibody and detection steps, and a concurrent control (general IHC practice); a single blank region does not establish CACYBP absence. |
💡Expected CACYBP appearanceA convincing positive shows clearly cell-associated, predominantly cytoplasmic staining in an HPA High population, such as esophageal squamous epithelial cells; isolated border staining or diffuse color without cell localization is suspect (HPA: esophagus IHC and tissue profile; UniProt Q9HB71: topology; general IHC practice).
| Cell population and reference intensity | HPA calls endocrine cells in appendix, colon, and duodenum High, but thyroid glandular cells Low (HPA: tissue IHC). Score the identified population and its compartment, since a whole-section average can conceal a sparse positive population (general IHC practice). |
| Antibody validation | HPA025753 and HPA057038 each have Enhanced IHC status (HPA: antibody validation). This supports the reported pattern, but does not prove specificity in every specimen or establish a dilution for the catalog antibody. |
| Topology and protein forms | CACYBP has no transmembrane segment, has three annotated isoforms, and lacks an annotated signal peptide or propeptide (UniProt Q9HB71: topology, isoforms, processing). Epitope coverage is unspecified here, so do not infer isoform-specific staining or a shed-protein pattern. |
| IF/ICC Q&A: Where should CACYBP appear? | Mainly in cytosol, with additional nucleoplasm staining (HPA: subcellular ICC-IF). UniProt also lists nucleus and cytoplasm, with calcium-dependent localization described in neuroblastoma cells (UniProt Q9HB71: subcellular location); that condition should not be assumed for paraffin tissue sections. |
| Situation | Likely cause | Next action |
|---|---|---|
| Known-positive endocrine or squamous cells have no chromogenic signal. | Tissue selection, an omitted reagent, weak detection, or retrieval conditions may explain a failed run (general IHC practice). | Confirm the named cell population and compare a concurrent positive section; then review antibody dilution, retrieval, and detection records (general IHC practice). Use HPA High cells as a reference, not as a guarantee of identical intensity in every section (HPA: tissue IHC). |
| Cardiomyocytes, myocytes, or fibroblasts stain strongly. | These populations are reported not detected (HPA: tissue IHC); cross-reactivity or endogenous detection activity is possible (general IHC practice). | Compare a reagent control, inspect whether color tracks the expected cytoplasmic cells, and repeat with a separately validated antibody if needed (general IHC practice; HPA: tissue profile and Enhanced antibody validation). |
| All cells show similar diffuse brown color. | A uniform deposit obscures the cell-specific pattern reported by HPA (HPA: tissue IHC). Nonspecific binding or excess detection signal is possible (general IHC practice). | Check the reagent control, blocking and wash steps, then adjust antibody or detection conditions using the concurrent positive section (general IHC practice). Reassess localization before scoring. |
| Staining appears only on membranes or tissue edges. | A membrane-dominant pattern is unexpected for a protein with no transmembrane segment and predominantly cytoplasmic tissue staining (UniProt Q9HB71: topology; HPA: tissue profile). Edge deposit may be a staining artifact (general IHC practice). | Inspect the section center and a reagent control, then review reagent distribution and wash coverage (general IHC practice). Require cell-associated cytoplasmic signal in an expected population before calling the section positive (HPA: tissue IHC). |
| A low-expression tissue gives a faint, uncertain signal. | Thyroid glandular cells, lung macrophages, and adipocytes are reported Low (HPA: tissue IHC); faint color alone may be hard to distinguish from background (general IHC practice). | Judge localization against the reagent control and include a High population, such as colon endocrine cells, in the run (HPA: colon and low-expression tissue IHC; general IHC practice). Record uncertainty rather than converting marginal color into a positive score. |
| A nuclear signal conflicts with the expected cytoplasmic tissue pattern. | Nuclear localization is reported, but tissue IHC is predominantly cytoplasmic (UniProt Q9HB71: subcellular location; HPA: tissue profile). Nuclear-only color may therefore need confirmation. | Check whether the same run shows cytoplasmic staining in an HPA High population and compare a reagent control (HPA: tissue IHC; general IHC practice). Describe confirmed nuclear staining separately from cytoplasmic intensity rather than merging compartments into one score (general IHC practice). |
Experiment evidence
IHC & IF images showing CACYBP staining
RP1104 · IHCIHC analysis of CACYBP using anti-CACYBP antibody (RP1104). CACYBP was detected in a paraffin-embedded section of human intestinal cancer ti
RP1104 · IHCIHC analysis of CACYBP using anti-CACYBP antibody (RP1104). CACYBP was detected in a paraffin-embedded section of mouse testis tissue. Heat
RP1104 · IFICC analysis of CACYBP using anti-CACYBP antibody (RP1104). CACYBP was detected in immunocytochemical section of MCF-7 cell. Enzyme antigen
PA1759 · IFIF analysis of CACYBP using anti-CACYBP antibody (PA1759). CACYBP was detected in an immunocytochemical section of Hela cells. Enzyme antige
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000116161-CACYBP/tissue/cerebral+cortex#imid_6690072
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000116161-CACYBP/tissue/colon#imid_6690006
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000116161-CACYBP/tissue/liver#imid_6689999
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000116161-CACYBP/tissue/kidney#imid_6690000
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA025753; green = CACYBP, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA025753; green = CACYBP, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for CACYBP IHC & IF
🧪Run colon first: its endocrine cells should stain (HPA: High in colon endocrine cells). Use skeletal muscle myocytes as the negative tissue (HPA: Not detected in skeletal muscle myocytes); on the colon slide, assess neighboring non-endocrine cells as potential internal negatives, but verify their status rather than assuming they lack CACYBP (HPA: High is assigned to endocrine cells).
Positive control tissue: Appendix (Endocrine cells, HPA High)
Negative control tissue: Heart muscle (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show CACYBP in A-431, U-251MG, U2OS, with annotated localisation: Cytosol (enhanced) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control; a concentration-matched rabbit IgG isotype control for the rabbit catalog antibody; and CACYBP knockout tissue or an antigen-peptide block where available (caption: rabbit anti-CACYBP RP1104; standard IHC practice). For chromogenic staining of colon, quench endogenous peroxidase and check endogenous biotin when using the caption’s biotin-based detection system (caption: biotinylated secondary and SABC; standard IHC practice).
⚠️Feasibility: The selected RP1104 paraffin-section caption reports EDTA retrieval at pH 8.0, but its fixative is unreported; no target-specific fixation window or fixation effect is supplied (caption: retrieval and fixative not stated). Treat that retrieval as a demonstrated starting condition for IHC, while the relative ease of frozen sections or IF remains unreported (caption: paraffin-section IHC only). For IF/ICC, cytosolic signal with additional nucleoplasmic signal is supported by HPA images, and colon tissue may require an autofluorescence control (HPA: subcellular ICC-IF; standard IF practice).
HPA tissue IHC evidence for CACYBP
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — 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 |
|---|---|---|---|---|
| Appendix | Endocrine cells | High | Protein (IHC) | HPA → |
| Cerebellum | Cells in molecular layer | High | Protein (IHC) | HPA → |
| Cerebral cortex | Neuronal cells | High | Protein (IHC) | HPA → |
| Colon | Endocrine cells | High | Protein (IHC) | HPA → |
| Duodenum | Endocrine cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Heart muscle | Cardiomyocytes | Not detected | Protein (IHC) | HPA → |
| Skeletal muscle | Myocytes | Not detected | Protein (IHC) | HPA → |
| Smooth muscle | Smooth muscle cells | Not detected | Protein (IHC) | HPA → |
| Soft tissue | Fibroblasts | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced CACYBP IHC Tips
Troubleshoot CACYBP staining in paraffin section IHC using the catalog antibody, with one companion answer for IF/ICC.
Which retrieval condition should I start with for CACYBP paraffin sections?
Start with heat-mediated antigen retrieval in EDTA at pH 8.0 (datasheet RP1104). The catalog image documents this condition in a paraffin-embedded human intestinal cancer section, followed by 1 μg/ml primary antibody overnight at 4°C (caption RP1104). If staining is weak, vary heating duration and cooling consistently across matched sections before considering another retrieval buffer as a fallback (standard IHC practice). Include a no-primary control and a reference section in each run so a retrieval change can be distinguished from background or run-to-run variation (standard IHC practice).
How should I troubleshoot weak staining when fixation history is uncertain?
The product caption identifies a paraffin-embedded section but does not state its fixative, so target-specific fixation sensitivity is unknown (caption RP1104). Record the fixative, fixation duration, tissue thickness and processing history for each specimen before comparing staining intensity (standard IHC practice). Keep retrieval at EDTA pH 8.0 and the initial primary concentration at 1 μg/ml while assessing sections with different documented fixation histories (datasheet RP1104; caption RP1104; standard IHC practice). Use a similarly processed reference section in each run, and treat a change associated with fixation history as a processing hypothesis requiring matched-sample confirmation (standard IHC practice).
Should CACYBP staining be cytoplasmic, nuclear, or both?
Expect predominantly cytoplasmic staining across many tissues, while assessing any nuclear signal separately (HPA tissue IHC: cytoplasmic expression in most tissues; HPA subcellular: cytosol and nucleoplasm). UniProt also lists nucleus and cytoplasm, with cytoplasmic localisation at low calcium and both compartments after retinoic acid induction and increased calcium in neuroblastoma cells (UniProt Q9HB71 localisation). Score nuclear and cytoplasmic staining independently within the same identified cell population, using consistent counterstain and thresholds (standard IHC practice). Do not infer a calcium-driven shift from a paraffin section alone; compare matched conditions and exclude overlapping cells or chromogen obscuring nuclear boundaries (UniProt Q9HB71 localisation; standard IHC practice).
Could isoforms or epitope masking explain discordant CACYBP staining?
CACYBP has 3 annotated isoforms and CS and SGS domains spanning residues 73–167 and 168–228, respectively (UniProt Q9HB71 isoforms and domains). The supplied product caption does not locate the antibody epitope or establish which isoforms it detects (caption RP1104). Therefore, do not assign differing tissue patterns to an isoform without epitope information and an independent isoform-specific measurement (UniProt Q9HB71 isoforms; standard IHC interpretation). If a known positive reference weakens after processing, repeat EDTA pH 8.0 retrieval under controlled heating and compare it with an independently validated antibody recognizing a documented region, when available (datasheet RP1104; standard IHC practice).
How can I check CACYBP localisation by multiplex IF/ICC?
For IF/ICC, pair CACYBP with a validated marker for the expected cell type, such as an endocrine-cell marker when examining appendix or colon, and inspect single channels before interpreting overlap (HPA tissue IHC: high in appendix and colon endocrine cells; standard IF practice). Choose fluorophores after checking specimen autofluorescence and reserve the cleaner channel for the weaker signal (standard IF practice). CACYBP lacks a transmembrane segment and is reported in cytosol and nucleoplasm, so permeabilisation must permit access to the intracellular compartment being assessed (UniProt Q9HB71 topology; HPA subcellular). Optimise permeabilisation against cell morphology and include single-primary and no-primary controls; the supplied caption documents IHC rather than IF conditions for RP1104 (caption RP1104; standard IF practice).
What should I adjust when DAB background obscures CACYBP staining?
The catalog workflow used 10% goat serum block, 1 μg/ml primary antibody overnight at 4°C, a biotinylated secondary for 30 minutes at 37°C, and SABC with DAB (caption RP1104). Check a no-primary section and reduce primary exposure or increase washing if diffuse color persists beyond expected cellular boundaries (standard IHC practice). Block endogenous peroxidase and evaluate endogenous biotin when using a biotin-based detection system, especially if signal remains in the no-primary section (caption RP1104; standard IHC practice). Compare background within the same tissue compartment and processing batch before lowering the primary concentration, since CACYBP is reported in many tissues (HPA tissue IHC; standard IHC practice).
How should I quantify CACYBP IHC across specimens? ⚠ ANSWER MARKED FOR VERIFICATION
Define cell populations before scoring, because HPA reports high signal in selected endocrine, neuronal and germinal center cells but no detection in several muscle cell types (HPA tissue IHC). For a specified population, record the percentage of positive cells and an H-score from intensity categories, scoring cytoplasmic and nuclear compartments separately (HPA subcellular; standard IHC practice). If spatial abundance matters, report positive-cell density per mm² of viable, annotated tissue alongside the total eligible-cell count (standard IHC practice). Normalise comparisons to the same cell type, viable area, section processing and staining run, with a common reference section to track batch variation (standard IHC practice).
How do I distinguish genuine CACYBP signal from staining artefact?
A plausible signal lies mainly in cytoplasm, with possible nuclear staining, and follows identifiable cells rather than an acellular deposit (HPA tissue IHC; HPA subcellular; standard IHC interpretation). Compare cell identity with the HPA pattern: appendix and colon endocrine cells show high staining, whereas cardiomyocytes and skeletal myocytes are listed as not detected (HPA tissue IHC). Treat staining restricted to section edges, necrotic regions or damaged tissue as suspect until it reproduces in intact areas and another section (standard IHC practice). Check no-primary and peroxidase-block controls for endogenous enzyme or detection background before calling unexpected compartments or cell types CACYBP-positive (standard IHC practice).
Boster reagents
Best CACYBP / Calcyclin-binding protein IHC Antibodies
Anti-CACYBP antibodies have IHC data from human intestinal cancer and mouse and rat testis paraffin sections, plus IF data from human HeLa cells (RP1104 and PA1759 image captions).
Real IHC data
Anti-SIAH Interacting Protein/CACYBP Antibody ®
Cat # RP1104
Real IF data
Anti-SIAH Interacting Protein/CACYBP Antibody ®
Cat # PA1759
RP1104 will render with an IHC figure from a human intestinal cancer paraffin section; its additional IHC captions show mouse and rat testis sections (RP1104 image captions). PA1759 will render with an IF figure from HeLa cells (PA1759 image caption).
Which to pick: Choose RP1104 for tissue IHC: it is listed for IHC in human, mouse and rat, with paraffin-section images in all three species (RP1104 applications, reactivity and IHC image captions). Choose PA1759 for IF/ICC when its HeLa IF example is useful; it is listed for IF and ICC in human, mouse and rat, but the supplied IF image shows human cells only (PA1759 applications, reactivity and IF image caption). For cross-species tissue IHC, RP1104 has the stronger direct evidence; its captions specify paraffin sections but do not report the fixative, and neither catalog entry reports clonality (RP1104 IHC image captions; catalog clone fields).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q9HB71 (CYBP_HUMAN, Calcyclin-binding protein).
- Human Protein Atlas. CACYBP tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. CACYBP subcellular location (ICC-IF): Mainly localized to the cytosol. In addition localized to the nucleoplasm..
- Human Protein Atlas. CACYBP antibody validation summary (2 antibodies).
- Evaluation of the Expression and Localization of the Multifunctional Protein CacyBP/SIP and Elements of the MAPK Signaling Pathway in the Adrenal Glands of Rats with Primary and Secondary Hypertension. International journal of molecular sciences 2023 — PMC10779320.
- Ageing-related changes in the levels of β-catenin, CacyBP/SIP, galectin-3 and immunoproteasome subunit LMP7 in the heart of men. PloS one 2020 — PMC7051089.
- Up-regulation of CacyBP/SIP during rat breast cancer development. Breast cancer (Tokyo, Japan) 2014 — PMC3996359.
- The Effect of CacyBP/SIP on the Phosphorylation of ERK1/2 and p38 Kinases in Clear Cell Renal Cell Carcinoma. International journal of molecular sciences 2023 — PMC10299707.
- PubMed PMID:12006993 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
- PubMed PMID:16710414 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for CACYBP (Calcyclin-binding protein). Expected localisation Tissue: mainly cytoplasmic (HPA tissue IHC); nuclear localisation is possible (UniProt). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet RP1104). Validated IHC antibody: RP1104 at 0.5-1μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Appendix (Endocrine cells); negative Heart muscle. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human intestinal cancer tissue; fixative not specified (datasheet RP1104); 2. Fixation: Image fixative and duration unreported (datasheet RP1104); 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 RP1104); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet RP1104); 8. Primary antibody: Rabbit anti-CACYBP, 0.5-1μg/ml (datasheet RP1104); 9. Primary incubation: Overnight at 4 °C (datasheet RP1104). Watch for: Fixation: Keep fixation consistent across sections. (standard IHC practice; not target-specific); Caveat: Cell-type mix can mask high endocrine or neuronal staining (HPA tissue IHC); Isoform / epitope: 3 isoforms; epitope coverage is unspecified (UniProt); Regulation: Expression regulation unannotated (UniProt). Evidence: UniProt Q9HB71, HPA tissue IHC · Enhanced, HPA ICC-IF · Mainly localized to the cytosol. In addition localized to the…, Datasheet RP1104, PMC10779320, PMC7051089. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/CACYBP/immunohistochemistry-ihc-guide.