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CNPY3 / Protein canopy homolog 3 · IHC design guide
Design Immunohistochemistry for CNPY3
Plan paraffin-section IHC for CNPY3 using the catalog antibody’s 1:50–1:200 dilution range (datasheet). Compare granular cytoplasmic staining with the tissue profile and use controls to assess the uncertain staining reliability (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
CNPY3 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 | Granular cytoplasm observed (HPA tissue IHC); ER expected (UniProt) | |
| Staining pattern | Granular cytoplasmic staining in most tissues (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen) | |
| Positive control | Bone marrow+4 more · see all | |
| Negative control | Adipose tissue+4 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 | Tissue staining has uncertain reliability (HPA tissue IHC) | |
| Regulation | Expression regulation not annotated (UniProt) | |
| Isoform / epitope | 2 isoforms; map the epitope, including signal peptide cleavage (UniProt) |
Section 1
Recommended CNPY3 IHC & IF Protocols
The catalog antibody protocol is followed by published CNPY3 IHC methods for colon cancer and breast cancer tissue microarrays (PMC11887163; PMC11271795; PMC12651230).
| Sample | Paraffin-embedded mouse kidney tissue; fixative not specified (datasheet A10971) |
| Fixation | Image fixative and duration unreported (datasheet A10971); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat-induced epitope retrieval in citrate buffer, pH 6.0, 20 min at 95–98 °C (standard rule: cytoplasmic / membrane antigen) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% normal serum of the secondary host, 30 min, room temperature (standard) |
| Primary antibody | Rabbit anti-CNPY3, 1:50-1:200 (datasheet A10971) |
| Primary incubation | Overnight at 4 °C (standard) |
| Detection | HRP-polymer secondary, DAB chromogen 5–10 min (standard) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | CNPY3-positive staining in hematopoietic cells of bone marrow (HPA tissue IHC: High). HPA tissue profile: Granular cytoplasmic expression in most tissues. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6.0 retrieval at 95–98 °C for 20 min (page antigen-retrieval rule); the breast cancer study also used sodium citrate (PMC12651230).
Section 2
What Is the Expected CNPY3 Staining Pattern?
CNPY3 is an endoplasmic reticulum protein without a transmembrane segment (UniProt Q9BT09 topology). In paraffin-section IHC, expect granular cytoplasmic staining, especially in bone marrow hematopoietic cells, fallopian tube glandular cells, and cerebellar molecular-layer cells (HPA: tissue IHC). Interpret the pattern cautiously: HPA rates tissue staining Uncertain because antibody staining and RNA expression have medium consistency and external verification is pending (HPA: tissue IHC).
| Granular cytoplasmic staining is strongest in bone marrow hematopoietic cells, fallopian tube glandular cells, or cerebellar molecular-layer cells (HPA: High in each). | This matches HPA's observed tissue profile and is compatible with endoplasmic reticulum localisation (HPA: tissue IHC; UniProt Q9BT09). Judge the named cells against adjacent tissue; HPA's Uncertain reliability means a matching pattern supports, but does not independently confirm, target specificity (HPA: tissue IHC). |
| Staining is predominantly nuclear, with little granular cytoplasmic signal in the expected cells. | A nuclear-dominant IHC result departs from HPA's granular cytoplasmic pattern and UniProt's endoplasmic reticulum annotation (HPA: tissue IHC; UniProt Q9BT09). Treat it as suspect and check controls and localisation before scoring it as CNPY3; the image alone cannot identify the artefact's cause. |
| Strong staining appears in adipocytes, ovarian stroma, or squamous epithelium while expected positive cells are weak (HPA: Not detected in those cell types). | That distribution conflicts with the reported IHC profile (HPA: tissue IHC). Cross-reactivity or endogenous chromogenic activity is possible; inspect the no-primary control and tissue morphology before assigning the signal to CNPY3 (general IHC practice). |
| Broad, diffuse colour covers cells and surrounding tissue, obscuring cytoplasmic granules. | Diffuse background prevents a reliable call against HPA's granular cytoplasmic reference pattern (HPA: tissue IHC). Check whether colour persists without primary antibody, then assess blocking, washing, primary concentration, and chromogen development as general IHC variables (general IHC practice). |
| No convincing signal appears in bone marrow hematopoietic cells or fallopian tube glandular cells (HPA: High in both). | A negative result in these reported positive cells makes the run difficult to interpret (HPA: tissue IHC). Check tissue preservation, retrieval and detection controls before calling biological absence (general IHC practice); HPA's Uncertain reliability also limits how definitive either reference tissue can be (HPA: tissue IHC). |
💡Expected CNPY3 appearanceA convincing IHC positive shows high, granular cytoplasmic staining in bone marrow hematopoietic or fallopian tube glandular cells (HPA: tissue IHC), whereas dominant nuclear colour or strong staining in HPA Not detected cells is suspect (HPA: tissue IHC; UniProt Q9BT09).
| Tissue and cell selection (HPA: tissue IHC) | HPA reports High staining in three cell groups: bone marrow hematopoietic, fallopian tube glandular, and cerebellar molecular-layer cells. It also reports Medium staining in several other cells, so intensity should be judged within the specified cell type rather than across a whole section (HPA: tissue IHC). |
| Subcellular reference (UniProt Q9BT09; HPA: tissue IHC) | UniProt places CNPY3 in the endoplasmic reticulum and reports no transmembrane segment; HPA describes granular cytoplasmic tissue staining. These sources support a cytoplasmic IHC expectation, but do not specify the exact shape or size of granules in a paraffin section. |
| Antibody evidence (HPA: antibody validation; tissue IHC) | HPA016560 is rated Uncertain for IHC and Approved for ICC, while the HPA tissue profile has Uncertain reliability. Keep scoring tied to cell type, compartment and controls; an ICC approval does not upgrade the IHC rating (HPA: antibody validation; tissue IHC). |
| Processing and isoforms (UniProt Q9BT09) | UniProt lists a signal peptide at residues 1–30, a mature chain at 31–278, one glycosylation site at residue 153, and two isoforms. The supplied sources give no antibody epitope or isoform coverage, so these features cannot predict which IHC signal will change with retrieval or processing. |
| IF/ICC Q&A: Should plasma membrane staining be expected? (HPA: subcellular ICC-IF) | HPA approves both endoplasmic reticulum and plasma membrane localisation in ICC-IF, with images from GAMG, OE19 and U2OS (HPA: subcellular ICC-IF). This is an IF/ICC observation; score paraffin-section IHC against its granular cytoplasmic tissue pattern instead (HPA: tissue IHC). |
| Situation | Likely cause | Next action |
|---|---|---|
| Reported High cells are unstained (HPA: tissue IHC). | The run may have inadequate antigen exposure or detection (general IHC practice); a blank slide alone does not establish absent CNPY3. | Check a reported positive cell group on the same run, then review retrieval, primary incubation and detection controls (HPA: tissue IHC; general IHC practice). |
| Colour is mainly nuclear. | Nuclear dominance conflicts with the expected endoplasmic reticulum and granular cytoplasmic pattern (UniProt Q9BT09; HPA: tissue IHC). | Confirm cell boundaries and inspect the no-primary control; withhold a CNPY3-positive call until cytoplasmic signal is resolved (general IHC practice). |
| Squamous cells or adipocytes stain strongly. | HPA reports these cells as Not detected; nonspecific binding or endogenous detection activity may contribute (HPA: tissue IHC; general IHC practice). | Compare the no-primary control, review blocking and detection chemistry, and rescore only interpretable cells (general IHC practice). |
| Diffuse colour hides the granular pattern. | Background from staining or detection conditions can obscure localisation (general IHC practice); HPA's reference pattern is granular cytoplasmic (HPA: tissue IHC). | Inspect the no-primary control, then review primary concentration, washes and chromogen development (general IHC practice). |
| Positive-cell intensity varies between sections. | HPA reports High and Medium levels in different named cell groups, and rates the tissue profile Uncertain (HPA: tissue IHC). | Compare the same cell type and compartment across sections, using consistent scoring criteria and run controls (HPA: tissue IHC; general IHC practice). |
| ICC-IF shows membrane signal, but IHC does not. | HPA approves plasma membrane and endoplasmic reticulum localisation for ICC-IF; its tissue IHC profile describes granular cytoplasmic staining (HPA: subcellular ICC-IF; tissue IHC). | Interpret each application against its own HPA reference and controls; do not require a visible membrane rim for an IHC-positive call (HPA: subcellular ICC-IF; tissue IHC). |
Experiment evidence
IHC & IF images showing CNPY3 staining
A10971 · IHCImmunohistochemistry of paraffin-embedded mouse kidney using CNPY3 antibody at dilution of 1:100 .Perform microwave antigen retrieval with 1
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000137161-CNPY3/tissue/cerebral+cortex#imid_4421512
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000137161-CNPY3/tissue/colon#imid_4421430
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000137161-CNPY3/tissue/liver#imid_4421439
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000137161-CNPY3/tissue/kidney#imid_4421432
HPA · ICC-IF GAMGHuman Protein Atlas immunofluorescence, GAMG, antibody HPA016560; green = CNPY3, blue = nucleus, red = microtubules.
HPA · ICC-IF GAMGHuman Protein Atlas immunofluorescence, GAMG, antibody HPA016560; green = CNPY3, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for CNPY3 IHC & IF
🧪Run fallopian tube first; glandular cells should stain (HPA: High in fallopian-tube glandular cells). Use cervix squamous epithelium as the negative tissue (HPA: Not detected in cervical squamous epithelial cells); compare non-glandular cells on the positive slide with background, but do not assume they are CNPY3-negative without validation (HPA: High designation applies to glandular cells).
Positive control tissue: Bone marrow (Hematopoietic cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show CNPY3 in GAMG, OE19, U2OS, with annotated localisation: Endoplasmic reticulum (approved), Plasma membrane (approved) (HPA subcellular).
Technical controls: Include a no-primary (secondary-only) slide, an isotype-matched antibody of the same host species and clonality, and CNPY3 knockout tissue or a validated peptide-block control (standard IHC control practice). For chromogenic detection, quench endogenous peroxidase and check background on the positive slide (standard IHC control practice).
⚠️Feasibility: A target-specific fixation window and fixation effect are unreported, and the selected A10971 paraffin-section caption does not state a fixative (datasheet: mouse-kidney IHC caption). That caption uses microwave retrieval in 10 mM PBS, pH 7.2, at 1:100, but does not establish that retrieval is required for other tissues (datasheet: A10971 mouse-kidney IHC caption). Whether frozen sections or IF are easier is unreported; assess tissue background during optimization (standard IHC practice).
HPA tissue IHC evidence for CNPY3
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Uncertain — Medium consistency between antibody staining and RNA expression data. Pending external verification.). 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 |
|---|---|---|---|---|
| Bone marrow | Hematopoietic cells | High | Protein (IHC) | HPA → |
| Cerebellum | Cells in molecular layer | High | Protein (IHC) | HPA → |
| Fallopian tube | Glandular cells | High | Protein (IHC) | HPA → |
| Adrenal gland | Glandular cells | Medium | Protein (IHC) | HPA → |
| Appendix | Glandular cells | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Cervix | Squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
| Esophagus | Squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
| Oral mucosa | Squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
| Ovary | Ovarian stroma cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced CNPY3 IHC Tips
Troubleshoot CNPY3 chromogenic IHC in paraffin sections by checking retrieval, compartment, cell identity, and controls before scoring.
How should I retrieve CNPY3 when paraffin sections stain weakly?
Start with citrate buffer at pH 6.0, heated to 95–98 °C for 20 minutes (page retrieval protocol). Allow sections to cool in retrieval buffer, then compare staining with a matched section processed without retrieval; keep antibody concentration and chromogen development identical so the comparison is interpretable. If staining remains weak, test microwave retrieval in 10 mM PBS, pH 7.2, as reported for paraffin-embedded mouse kidney with antibody A10971 at 1:100 (A10971 tissue-IHC caption). Because CNPY3 is an endoplasmic reticulum protein (UniProt Q9BT09 subcellular annotation), judge improvement by intracellular signal and preservation of cell detail, not by darker section edges.
Could fixation be hiding CNPY3 in my paraffin sections?
Target-specific fixation sensitivity is unknown: the A10971 paraffin-section caption does not state a fixative (A10971 tissue-IHC caption). For a diagnostic comparison, process sections from documented fixation conditions together and keep retrieval, antibody incubation, and chromogen development constant; record fixation time rather than assuming that weak staining proves epitope damage. Inspect morphology after retrieval, since loss of tissue detail or detached sections can make apparent staining differences difficult to interpret. CNPY3 has a signal peptide, a glycosylation site at residue 153, and no transmembrane segment (UniProt Q9BT09 sequence annotation), but those features do not establish a preferred fixative or a target-specific fixation effect.
Where should convincing CNPY3 staining appear in tissue?
Expect chiefly granular cytoplasmic staining in tissue sections (HPA tissue-IHC profile), consistent with endoplasmic reticulum localisation (UniProt Q9BT09 subcellular annotation). HPA subcellular imaging also assigns CNPY3 to the plasma membrane (HPA subcellular summary), so evaluate membrane staining alongside intracellular signal rather than treating either pattern as definitive by itself. Compare the signal with cell morphology and a matched negative control, paying attention to whether granules lie within intact cells instead of along section folds or torn edges. HPA rates its tissue-IHC reliability as uncertain and pending external verification (HPA tissue-IHC reliability), so use independent biological or antibody controls before assigning a new compartment.
Could isoforms or processing explain inconsistent CNPY3 staining?
CNPY3 has 2 annotated isoforms, and its precursor carries a signal peptide at residues 1–30 with a processed chain spanning 31–278 (UniProt Q9BT09 sequence annotation). The supplied IHC caption does not identify the A10971 epitope (A10971 tissue-IHC caption), so do not assume that staining distinguishes isoforms or recognizes the cleaved signal peptide. Ask for the immunogen or epitope coordinates before interpreting a negative section as isoform loss, and check whether those coordinates occur in the isoform under study. If staining varies between otherwise comparable sections, first compare retrieval and tissue preservation under identical detection conditions; epitope accessibility remains a hypothesis until tested directly.
How should I check CNPY3 by multiplex immunofluorescence?
For a secondary IF experiment, pair CNPY3 with a marker of the expected cell type: hematopoietic cells in bone marrow are reported high for CNPY3 (HPA tissue-IHC profile), although that tissue evidence does not validate the IF assay. Choose spectrally separated fluorophores and place the weaker channel away from the tissue's strongest autofluorescence; inspect single-label and unstained controls before interpreting overlap. Permeabilise sufficiently to access an endoplasmic reticulum epitope (UniProt Q9BT09 subcellular annotation), while recognising that the catalog antibody's epitope and membrane-facing side are not specified in the supplied caption (A10971 tissue-IHC caption). Optimise permeabilisation empirically and retain an unpermeabilised comparison if apparent plasma membrane signal is central to the claim (HPA subcellular summary).
How can I reduce diffuse brown staining without losing CNPY3 signal?
Start with a no-primary control, a suitable protein block, and a peroxidase block; these are general chromogenic IHC controls rather than CNPY3-specific conditions. Titrate the primary antibody and shorten chromogen development while keeping retrieval fixed, then inspect whether intracellular granules remain distinguishable from diffuse haze. The A10971 mouse-kidney caption reports 1:100 antibody dilution and microwave retrieval in 10 mM PBS, pH 7.2 (A10971 tissue-IHC caption); use those as a documented comparison, not a universal dilution. Examine pigment, tissue folds, necrosis, and section edges separately, because a brown deposit outside intact cells cannot establish the granular cytoplasmic pattern reported for CNPY3 (HPA tissue-IHC profile).
What should I score when CNPY3 staining varies across cells? ⚠ ANSWER MARKED FOR VERIFICATION
Define the analysis compartment and cell population before scoring, using granular cytoplasmic staining as the tissue pattern to assess (HPA tissue-IHC profile). Record either an H-score from intensity and percentage of positive cells, or the percentage of positive cells alone; for sparse populations, report positive-cell density per mm² of viable tissue. Normalise counts to the number of eligible cells or the measured viable area, and compare sections stained in the same run with identical retrieval and chromogen development. Report cell types separately because HPA lists high staining in bone-marrow hematopoietic cells but no detection in adipocytes (HPA tissue-IHC profile), and flag the HPA tissue-IHC reliability as uncertain (HPA tissue-IHC reliability).
When is brown staining convincing evidence of CNPY3?
A convincing result places granular cytoplasmic signal within intact, relevant cells (HPA tissue-IHC profile) and is compatible with endoplasmic reticulum localisation (UniProt Q9BT09 subcellular annotation). Compare a positive population such as bone-marrow hematopoietic cells with a population reported as undetected, such as adipocytes, while recognising that these HPA tissue observations have uncertain reliability (HPA tissue-IHC profile and reliability). Treat staining confined to necrosis, cut edges, folds, or the no-primary control as suspect; use a peroxidase block to check whether endogenous enzyme activity contributes to chromogen deposition. Predominantly nuclear staining needs independent validation because the supplied localisation evidence identifies endoplasmic reticulum and plasma membrane, not nucleus (UniProt Q9BT09 subcellular annotation; HPA subcellular summary).
Boster reagents
Best CNPY3 / Protein canopy homolog 3 IHC Antibodies
A10971 is listed for IHC with human and mouse reactivity (catalog: applications/reactivity). Its IHC figure shows paraffin-embedded mouse kidney; the fixative is unreported (A10971 image caption).
Real IHC data
Anti-Protein canopy homolog 3 CNPY3 Antibody
Cat # A10971
A10971 is listed for IHC and WB with human and mouse reactivity (catalog: applications/reactivity). Its IHC figure shows paraffin-embedded mouse kidney stained at 1:100 after microwave retrieval in 10 mM PBS, pH 7.2 (A10971 image caption).
Which to pick: For tissue IHC, choose A10971 for paraffin sections; its own image documents mouse kidney, and its listed IHC range is 1:50–1:200 (A10971 image caption; datasheet: IHC dilution). There is no IF/ICC pick because A10971 has neither an IF/ICC application listing nor an IF image (catalog: applications/IF images). A10971 is the cross-species candidate based on listed human and mouse reactivity, though its IHC image documents mouse tissue only; the fixative is unreported (catalog: reactivity; A10971 image caption).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q9BT09 (CNPY3_HUMAN, Protein canopy homolog 3).
- Human Protein Atlas. CNPY3 tissue IHC expression (reliability: Uncertain).
- Human Protein Atlas. CNPY3 subcellular location (ICC-IF): Mainly localized to the plasma membrane and endoplasmic reticulum..
- Human Protein Atlas. CNPY3 antibody validation summary (1 antibodies).
- CNPY3's regulation of tumor microenvironment and its impact on colon cancer aggressiveness. Molecular medicine (Cambridge, Mass.) 2025 — PMC11887163.
- Canopy FGF signaling regulator 3 affects prognosis, immune infiltration, and PI3K/AKT pathway in colon adenocarcinoma. World journal of gastrointestinal oncology 2024 — PMC11271795.
- CNPY3 Promotes Human Breast Cancer Progression and Metastasis via Modulation of the Tumor Microenvironment. Current issues in molecular biology 2025 — PMC12651230.
- RNA-Binding Protein HuR Regulates Paneth Cell Function by Altering Membrane Localization of TLR2 via Post-transcriptional Control of CNPY3. Gastroenterology 2019 — PMC6707881.
- PubMed PMID:12975309 — UniProt-cited evidence.
- PubMed PMID:14574404 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for CNPY3 (Protein canopy homolog 3). Expected localisation Granular cytoplasm observed (HPA tissue IHC); ER expected (UniProt). Antigen retrieval: Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen). Validated IHC antibody: A10971 at 1:50-1:200 (the product's own IHC validation image is shown). Controls (HPA): positive Bone marrow (Hematopoietic cells); negative Adipose tissue. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Paraffin-embedded mouse kidney tissue; fixative not specified (datasheet A10971); 2. Fixation: Image fixative and duration unreported (datasheet A10971); 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-induced epitope retrieval in citrate buffer, pH 6.0 , 20 min at 95–98 °C…; 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% normal serum of the secondary host, 30 min , room temperature (standard); 8. Primary antibody: Rabbit anti-CNPY3, 1:50-1:200 (datasheet A10971); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific); Caveat: Tissue staining has uncertain reliability (HPA tissue IHC); Isoform / epitope: 2 isoforms; map the epitope, including signal peptide cleavage (UniProt); Regulation: Expression regulation not annotated (UniProt). Evidence: UniProt Q9BT09, HPA tissue IHC · Uncertain, HPA ICC-IF · Mainly localized to the plasma membrane and endoplasmic reticulum., Datasheet A10971, PMC11887163, PMC11271795. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/CNPY3/immunohistochemistry-ihc-guide.