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CALCOCO2 / Calcium-binding and coiled-coil domain-containing protein 2 · IHC design guide
Design Immunohistochemistry for CALCOCO2
Plan chromogenic IHC for CALCOCO2 in paraffin sections with the IHC-validated antibody at 0.5–1 μg/mL (datasheet A05876-1). Assess cytoplasmic staining and account for variation among tissues and cell types (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
CALCOCO2 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 | Variable cytoplasmic tissue staining (HPA tissue IHC) | |
| Staining pattern | Cytoplasmic staining at variable levels across cell types (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A05876-1) | |
| Positive control | Appendix+4 more · see all | |
| Negative control | Soft tissue+2 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep paraffin-section fixation consistent (standard IHC practice; not target-specific) | |
| Caveat | Staining has medium agreement with RNA expression (HPA tissue IHC) | |
| Regulation | Staining varies by tissue (HPA tissue IHC) | |
| Isoform / epitope | 5 isoforms; check antibody epitope coverage (UniProt) |
Section 1
Recommended CALCOCO2 IHC & IF Protocols
Compare the catalog antibody’s EDTA pH 8.0 IHC-P protocol (datasheet: A05876-1) with the published mouse coronary artery IHC protocol (PMC13242735).
| Sample | Paraffin-embedded human lung cancer tissue; fixative not specified (datasheet A05876-1) |
| Fixation | Image fixative and duration unreported (datasheet A05876-1); 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 A05876-1); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A05876-1) |
| Primary antibody | Rabbit anti-CALCOCO2, 0.5-1μg/ml (datasheet A05876-1) |
| Primary incubation | Overnight at 4 °C (datasheet A05876-1) |
| Detection | Streptavidin-biotin complex (SABC), DAB chromogen (datasheet A05876-1) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | CALCOCO2-positive staining in glandular cells of appendix (HPA tissue IHC: Medium). HPA tissue profile: Cytoplasmic expression at variable levels. No signal in the no-primary control. |
💡Decision noteStart with EDTA pH 8.0 heat retrieval for the catalog antibody (datasheet: A05876-1); evaluate citrate pH 6.0 retrieval for the published protocol (PMC13242735).
Section 2
What Is the Expected CALCOCO2 Staining Pattern?
CALCOCO2 should appear mainly in the cytoplasm, sometimes concentrated near the nucleus or associated with vesicles (UniProt Q13137 subcellular location; HPA subcellular). In paraffin-section IHC, expect variable cytoplasmic staining in the cell populations documented by HPA, including glandular, endothelial and hematopoietic cells (HPA tissue IHC). HPA rates the tissue staining Enhanced, with medium consistency between staining and RNA expression (HPA tissue IHC). CALCOCO2 has no transmembrane segment (UniProt Q13137 topology).
| Medium cytoplasmic staining in appendix or duodenal glandular cells. | This matches documented positive cell populations and levels (HPA tissue IHC). Score the glandular cells separately from adjacent cells; HPA reports variable cytoplasmic expression, so a uniformly intense field is not the expected reference pattern (HPA tissue IHC). |
| Medium cytoplasmic staining in cerebral-cortex or colon endothelial cells, or bone-marrow hematopoietic cells. | These are additional documented positive populations (HPA tissue IHC). Identify the stained cell type before calling the tissue positive: the HPA observations apply to specified cells within each tissue, not every cell in the section (HPA tissue IHC). |
| Predominantly nuclear or sharply membrane-rimmed staining, with little cytoplasmic signal. | Recheck compartment assignment and staining controls. The supported pattern is cytoplasmic, with perinuclear and vesicle-associated localization (UniProt Q13137 subcellular location; HPA subcellular). UniProt notes conflicting historical reports of nuclear dots, so isolated nuclear puncta alone are inconclusive (UniProt Q13137 subcellular location). |
| Staining in cells listed as not detected, or a uniform haze across many cell types. | Fibroblasts in soft tissue, red-pulp cells in spleen and vaginal squamous cells were not detected in the supplied HPA observations (HPA tissue IHC). Strong staining there warrants checks for cross-reactivity or endogenous detection activity; diffuse haze also warrants a background-control check (general IHC practice). |
| No staining in a documented positive cell population. | A negative result in appendix glandular cells, for example, conflicts with their reported medium staining (HPA tissue IHC). Confirm that the relevant cells are present, then inspect the run controls and assay setup before interpreting the result as absent CALCOCO2 (general IHC practice). |
💡Expected CALCOCO2 appearanceCall a result positive when the appropriate cells show variable, typically medium cytoplasmic staining, possibly perinuclear; dominant nuclear or indiscriminate staining needs review (HPA tissue IHC; UniProt Q13137 subcellular location).
| Cell type and tissue context | HPA reports medium staining in several specified glandular, endothelial and hematopoietic populations, but low or undetected staining in others (HPA tissue IHC). Compare matching cell types rather than whole-section darkness. |
| Compartment and topology | CALCOCO2 is cytoplasmic and can associate with autophagosome membranes; it has no transmembrane segment (UniProt Q13137 subcellular location and topology). A crisp plasma-membrane outline alone is therefore an unsupported assignment. |
| Strength of evidence | Enhanced is HPA's IHC reliability rating; its description still reports medium consistency between antibody staining and RNA expression (HPA tissue IHC). Treat an unexpected pattern as a finding to check, not a new reference pattern. |
| Isoforms and antibody comparison | UniProt lists five CALCOCO2 isoforms, while the supplied records give no antibody epitope coverage (UniProt Q13137 isoforms; HPA antibodies). HPA lists three rabbit polyclonal antibodies with Enhanced IHC status; comparison may help assess a disputed pattern (HPA antibodies). |
| Q: What should IF/ICC show? | A: Mainly cytosol, with additional vesicles (HPA subcellular). HPA marks cytosol Supported and vesicles Approved; its image-listed cell lines are A-431, U-251MG and U2OS (HPA subcellular). This localization check does not specify an IF/ICC protocol. |
| Situation | Likely cause | Next action |
|---|---|---|
| The slide shows mainly nuclear dots. | Historical nuclear-dot localization reports conflict with reports of predominantly coarse, perinuclear cytoplasm (UniProt Q13137 subcellular location). | Check the cytoplasmic pattern in a documented positive cell population and compare with staining controls before assigning the nuclear signal to CALCOCO2 (HPA tissue IHC; general IHC practice). |
| A reported negative population stains strongly. | Unexpected staining may reflect antibody cross-reactivity or endogenous detection activity (general IHC practice); HPA lists specific undetected populations, not an absolute rule for every specimen (HPA tissue IHC). | Verify cell identity; inspect no-primary and detection controls, then compare another IHC-validated antibody if available (general IHC practice; HPA antibodies). |
| The entire section has diffuse brown background. | Broad haze obscures the cell-specific cytoplasmic pattern reported by HPA and can arise from assay background (HPA tissue IHC; general IHC practice). | Review no-primary and detection controls, blocking, washes and chromogen exposure; assess the signal in identified cells after background is resolved (general IHC practice). |
| A known-positive population is blank. | The relevant cells may be absent from the sampled area, or the IHC run may have failed (general IHC practice); HPA reports medium staining in several specified positive populations (HPA tissue IHC). | Confirm tissue morphology and the expected cells, inspect run controls, then review retrieval and antibody conditions using the validated IHC workflow (general IHC practice). |
| Only a sharp cell-surface rim is visible. | This differs from HPA's cytoplasmic tissue profile and UniProt's lack of a transmembrane segment (HPA tissue IHC; UniProt Q13137 topology). | Check section edges, counterstain and detection controls; require convincing cytoplasmic staining in an appropriate cell type before scoring it as CALCOCO2 (general IHC practice; HPA tissue IHC). |
| Results differ between tissues or sections. | HPA reports variable cytoplasmic expression and low tissue RNA specificity; its Enhanced rating has medium staining-to-RNA consistency (HPA tissue IHC). Variation alone does not identify a technical fault. | Compare the same annotated cell population across sections, record localization and intensity separately, and use run controls to investigate any unexplained loss of signal (HPA tissue IHC; general IHC practice). |
Experiment evidence
IHC & IF images showing CALCOCO2 staining
A05876-1 · IHCIHC analysis of NDP52/CALCOCO2 using anti-NDP52/CALCOCO2 antibody (A05876-1). NDP52/CALCOCO2 was detected in paraffin-embedded section of hu
A05876-1 · IHCIHC analysis of NDP52/CALCOCO2 using anti-NDP52/CALCOCO2 antibody (A05876-1). NDP52/CALCOCO2 was detected in paraffin-embedded section of hu
A05876-1 · IFIF analysis of NDP52/CALCOCO2 using anti-NDP52/CALCOCO2 antibody (A05876-1). NDP52/CALCOCO2 was detected in immunocytochemical section of He
M05876-2 · IFIF analysis of NDP52/CALCOCO2 using anti-NDP52/CALCOCO2 antibody (M05876-2). NDP52/CALCOCO2 was detected in an immunocytochemical section of
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000136436-CALCOCO2/tissue/cerebral+cortex#imid_5905248
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000136436-CALCOCO2/tissue/colon#imid_5905174
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000136436-CALCOCO2/tissue/liver#imid_5905695
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000136436-CALCOCO2/tissue/kidney#imid_5905680
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA022989; green = CALCOCO2, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA022989; green = CALCOCO2, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for CALCOCO2 IHC & IF
🧪Run appendix first and look for staining in its glandular cells (HPA: Appendix, glandular cells, Medium). Use soft-tissue fibroblasts as a negative tissue (HPA: Soft tissue, fibroblasts, Not detected); on the appendix slide, treat adjacent unstained nonglandular cells as background references only if they are present, rather than assuming they are negative.
Positive control tissue: Appendix (Glandular cells, HPA Medium)
Negative control tissue: Soft tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show CALCOCO2 in A-431, U-251MG, U2OS, with annotated localisation: Cytosol (supported) (HPA subcellular).
Technical controls: Include a no-primary (secondary-only) control, a nonimmune rabbit IgG control matched to the primary's concentration and clonality where applicable, and CALCOCO2-knockout material as a biological negative (caption: rabbit primary; standard IHC practice). With the caption's biotinylated secondary and SABC/DAB detection, check endogenous peroxidase and biotin background in appendix sections (caption: biotinylated secondary and SABC/DAB; HPA: Appendix glandular cells).
⚠️Feasibility: A CALCOCO2-specific fixation window and the effect of fixation are unreported; the selected A05876-1 paraffin-section caption does not state a fixative (caption: fixative not stated). That caption uses heat-mediated EDTA retrieval at pH 8.0, but does not establish that retrieval is required (caption: EDTA pH 8.0). The supplied evidence does not establish whether frozen sections or IF are easier; in appendix, distinguish intracellular glandular staining from luminal deposits when scoring (HPA: Appendix glandular cells; standard IHC practice).
HPA tissue IHC evidence for CALCOCO2
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 | Glandular cells | Medium | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | Medium | Protein (IHC) | HPA → |
| Cerebral cortex | Endothelial cells | Medium | Protein (IHC) | HPA → |
| Colon | Endothelial cells | Medium | Protein (IHC) | HPA → |
| Duodenum | Glandular cells | Medium | Protein (IHC) | HPA → |
Section 3
Advanced CALCOCO2 IHC Tips
Troubleshoot CALCOCO2 staining in paraffin section IHC using the catalog antibody’s tissue image and independent localisation evidence.
Which retrieval conditions should I start with for CALCOCO2 paraffin sections?
Start with heat mediated retrieval in EDTA, pH 8.0, for paraffin sections (datasheet A05876-1). The documented CALCOCO2 tissue image used that retrieval before overnight incubation at 4°C with 1 µg/ml primary antibody (datasheet A05876-1). If staining is weak, adjust heating duration on matched sections while holding the buffer and detection conditions constant; inspect morphology alongside signal because excessive retrieval can damage sections (general IHC practice). Compare each adjustment with a no primary control and a section processed under the documented conditions, then judge improvement in cytoplasmic staining within intact cells (HPA tissue IHC: variable cytoplasmic expression).
How should I troubleshoot weak staining when fixation details are missing?
Treat CALCOCO2 sensitivity to fixation as unknown: the selected paraffin section caption does not state a fixative (datasheet A05876-1). Record the specimen’s actual fixative, fixation duration and processing history before comparing staining across cases; differences in these steps can alter antigen accessibility in routine IHC (general IHC practice). Keep retrieval at EDTA, pH 8.0 and the documented primary concentration at 1 µg/ml while assessing matched sections (datasheet A05876-1). If a newly processed section stains differently, check tissue preservation and run a known staining control in the same batch before assigning the difference to CALCOCO2 abundance (general IHC practice).
Where should CALCOCO2 staining appear, and how should nuclear signal be handled?
Expect predominantly cytoplasmic staining, often near the nucleus, because UniProt places CALCOCO2 in the cytoplasm and perinuclear region and HPA reports variable cytoplasmic tissue expression (UniProt Q13137 subcellular location; HPA tissue IHC). Vesicular or coarse cytoplasmic patterns can also fit its reported autophagosome membrane association and supported cytosolic localisation with additional vesicles (UniProt Q13137 subcellular location; HPA subcellular). Score cytoplasmic signal separately from any nuclear staining and compare both with a no primary control on adjacent sections (general IHC practice). Historical nuclear dot reports conflict with later predominantly cytoplasmic reports, so isolated nuclear dots need independent confirmation before assignment to CALCOCO2 (UniProt Q13137 subcellular location).
Could isoforms or epitope accessibility explain uneven CALCOCO2 staining?
CALCOCO2 has 5 listed isoforms, so confirm which sequence region the antibody recognizes before interpreting absent staining as absent protein (UniProt Q13137 isoforms). The supplied tissue caption documents staining conditions but gives no epitope map or isoform coverage for A05876-1 (datasheet A05876-1). Compare matched sections using the documented EDTA, pH 8.0 retrieval and 1 µg/ml primary concentration before changing one variable at a time (datasheet A05876-1). CALCOCO2 has no annotated transmembrane segment and has a reported phosphoserine at residue 445, but neither fact establishes that fixation or retrieval masks this antibody’s epitope (UniProt Q13137 topology and modified residues).
How can IF help check an ambiguous CALCOCO2 IHC pattern?
Use IF as a separate localisation check, with a marker identifying the cell population being assessed in the IHC section; HPA reports medium cytoplasmic staining in appendix glandular cells and colon endothelial cells (HPA tissue IHC). Choose spectrally separated fluorophores and place a weak CALCOCO2 signal in a channel with low measured tissue autofluorescence, using unstained and single label controls to set the readout (general IF practice). CALCOCO2 is mainly cytosolic, with additional vesicular localisation and no transmembrane segment, so test permeabilisation that gives antibody access to cytoplasmic epitopes without losing cell structure (HPA subcellular; UniProt Q13137 topology). Compare the resulting cell and compartment pattern with chromogenic IHC, while treating IF conditions as independently optimised (general IF practice).
What controls help separate CALCOCO2 signal from chromogenic background?
Run a no primary control through secondary antibody, detection and chromogen steps to reveal nonspecific or endogenous colour development (general IHC practice). The documented tissue image used 10% goat serum, biotinylated goat anti rabbit secondary antibody, a streptavidin biotin complex and DAB (datasheet A05876-1). For that detection scheme, assess endogenous peroxidase and biotin background with appropriate controls before raising primary antibody concentration; apply a peroxidase block as a general chromogenic IHC step (general IHC practice). Review staining at tissue edges, damaged areas and necrotic regions separately from intact cellular cytoplasm, which is the reported tissue pattern for CALCOCO2 (HPA tissue IHC; general IHC practice).
How should I quantify variable CALCOCO2 staining across sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and cytoplasmic compartment before scoring, because HPA describes variable cytoplasmic CALCOCO2 expression across tissues (HPA tissue IHC). For each region, record the percentage of positive intact cells and an H-score from intensity categories, or count positive cells per mm² when cell density is the main endpoint (general IHC quantification practice). Normalise counts to the evaluated tissue area or eligible cell count, and use the same threshold, counterstain assessment and imaging settings across cases (general IHC quantification practice). Report cell types separately where appropriate: HPA lists medium staining in appendix glandular cells and bone marrow hematopoietic cells, but no detection in soft tissue fibroblasts (HPA tissue IHC).
When is a CALCOCO2 positive result biologically credible?
A credible result places staining in intact cell cytoplasm, potentially near the nucleus or in vesicular structures, consistent with the reported CALCOCO2 distribution (UniProt Q13137 subcellular location; HPA subcellular; HPA tissue IHC). Check that the stained cell population matches the region being scored: HPA reports medium staining in duodenal glandular cells and no detection in spleen red pulp cells (HPA tissue IHC). Treat isolated nuclear staining cautiously because nuclear dot localisation reports conflict with later predominantly cytoplasmic findings (UniProt Q13137 subcellular location). Edge staining, necrotic deposits or colour persisting in a no primary control warrant investigation of section artefact or endogenous enzyme activity before interpreting biological expression (general IHC practice).
Boster reagents
Best CALCOCO2 / Calcium-binding and coiled-coil domain-containing protein 2 IHC Antibodies
The catalog shows CALCOCO2 staining in human paraffin sections and cultured cells (catalog image captions). Listed reactivity is human for A05876-1 and human, mouse and rat for M05876-2 (catalog reactivity).
Real IHC data
Anti-NDP52/CALCOCO2 Antibody ®
Cat # A05876-1
Real IF data
Anti-NDP52/CALCOCO2 Antibody ® (monoclonal, 9E2F2)
Cat # M05876-2
A05876-1 has IHC images from paraffin-embedded human lung and mammary cancer tissue and an IF image from HeLa cells (A05876-1 image captions). M05876-2 has an IF/ICC image from U20S cells (M05876-2 image caption).
Which to pick: Choose A05876-1 for chromogenic paraffin-section IHC: its captions show EDTA retrieval at pH 8.0 and DAB detection; the fixative is unreported (A05876-1 IHC captions). For IF/ICC, both SKUs list those applications (catalog application lists); M05876-2 is the monoclonal option, clone 9E2F2 (catalog clone). Choose M05876-2 when listed human, mouse and rat reactivity is needed for IF/ICC, while A05876-1 lists human reactivity (catalog applications/reactivity).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q13137 (CACO2_HUMAN, Calcium-binding and coiled-coil domain-containing protein 2).
- Human Protein Atlas. CALCOCO2 tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. CALCOCO2 subcellular location (ICC-IF): Mainly localized to the cytosol. In addition localized to vesicles..
- Human Protein Atlas. CALCOCO2 antibody validation summary (3 antibodies).
- Multi-omics data mining combined with experimental validation reveals ferroptosis- and autophagy-associated hub genes as diagnostic candidates and immune modulators in atherosclerosis. Animal models and experimental medicine 2026 — PMC13242735.
- Mechanistic insights into COVID-19 by global analysis of the SARS-CoV-2 3CL(pro) substrate degradome. Cell reports 2021 — PMC8501228.
- PubMed PMID:7540613 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
- PubMed PMID:16625196 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for CALCOCO2 (Calcium-binding and coiled-coil domain-containing protein 2). Expected localisation Variable cytoplasmic tissue staining (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A05876-1). Validated IHC antibody: A05876-1 at 0.5-1μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Appendix (Glandular cells); negative Soft tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human lung cancer tissue; fixative not specified (datasheet A05876-1); 2. Fixation: Image fixative and duration unreported (datasheet A05876-1); 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 A05876-1); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A05876-1); 8. Primary antibody: Rabbit anti-CALCOCO2, 0.5-1μg/ml (datasheet A05876-1); 9. Primary incubation: Overnight at 4 °C (datasheet A05876-1). Watch for: Fixation: Keep paraffin-section fixation consistent (standard IHC practice; not target-specific); Caveat: Staining has medium agreement with RNA expression (HPA tissue IHC); Isoform / epitope: 5 isoforms; check antibody epitope coverage (UniProt); Regulation: Staining varies by tissue (HPA tissue IHC). Evidence: UniProt Q13137, HPA tissue IHC · Enhanced, HPA ICC-IF · Mainly localized to the cytosol. In addition localized to vesicles., Datasheet A05876-1, PMC13242735. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/CALCOCO2/immunohistochemistry-ihc-guide.