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CGN / Cingulin · IHC design guide
Design Immunohistochemistry for CGN
Plan CGN staining in paraffin sections with the IHC-validated antibody at 1:50–1:200 (datasheet: A02373). Use high-staining epithelial tissue as a positive control and assess cytoplasmic and membranous signal in light of CGN’s expected junctional location (HPA tissue IHC; UniProt).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
CGN 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 | Cytoplasmic and membranous staining (HPA tissue IHC); apical junction expected (UniProt) | |
| Staining pattern | Epithelia show cytoplasmic and membranous staining (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen) | |
| Positive control | Appendix+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 formalin fixation consistent across sections. (standard IHC practice; not target-specific) | |
| Caveat | Antibody staining and RNA show medium consistency (HPA tissue IHC) | |
| Regulation | Intestine-enhanced RNA expression (HPA tissue RNA) | |
| Isoform / epitope | 2 isoforms; check epitope coverage (UniProt) |
Section 1
Recommended CGN IHC & IF Protocols
The catalog antibody’s IHC-P protocol is followed by published CGN staining methods for human lung tissue (PMC10855320), dog skin (PMC13314070), and salivary duct adenocarcinoma tissue (PMC9179926).
| Sample | Paraffin-embedded Human colon carcinoma tissue; fixative not specified (datasheet A02373) |
| Fixation | Image fixative and duration unreported (datasheet A02373); 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-CGN, 1:50-1:200 (datasheet A02373) |
| 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 | CGN-positive staining in enterocytes - Microvilli of appendix (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic and membranous expression in several tissues, most abundant in gastrointestinal tract. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6.0 retrieval at 95–98 °C for 20 min (page retrieval); use ER2 autoclave retrieval when reproducing the lung or salivary protocols (PMC10855320; PMC9179926).
Section 2
What Is the Expected CGN Staining Pattern?
CGN is a junctional protein on the cytoplasmic face of tight junctions in polarized epithelia and some endothelia; it has no transmembrane segment (UniProt Q9P2M7). In paraffin-section IHC, expect staining in selected epithelial cells, especially gastrointestinal epithelium (HPA tissue IHC). HPA describes cytoplasmic and membranous staining, with Enhanced reliability but only medium consistency between antibody staining and RNA expression (HPA tissue IHC).
| Apical-border staining in colon or duodenal enterocytes, with some cytoplasmic signal. | This fits high enterocyte staining and HPA’s cytoplasmic and membranous profile (HPA tissue IHC). Junction-associated enrichment fits CGN’s location on the cytoplasmic face of epithelial tight junctions (UniProt Q9P2M7). The tissue image alone does not resolve every junction. |
| Strong, isolated nuclear staining dominates otherwise positive epithelium. | A nuclear-dominant pattern is discordant with the reported cell-junction location and HPA membrane/cell-junction ICC-IF localization (UniProt Q9P2M7; HPA subcellular). Treat it as suspect; review controls and detection before assigning it to CGN. |
| Prominent staining appears in an HPA-negative cell population or across unrelated cells. | That distribution needs scrutiny for antibody cross-reactivity or endogenous detection activity (general IHC practice). HPA reports no detection in adipocytes or bone-marrow hematopoietic cells, for example; those findings apply to the specified cells, not every cell in those tissues (HPA tissue IHC). |
| Uniform haze covers cells and surrounding tissue, obscuring cell borders. | The haze does not show the expected cell-linked distribution (UniProt Q9P2M7; HPA tissue IHC). Nonspecific antibody binding, incomplete blocking, or chromogen background are possible general IHC causes; use controls to distinguish them (general IHC practice). |
| No signal appears in colon enterocytes or kidney collecting ducts. | Both are reported as High in the named cell populations (HPA tissue IHC). A blank run therefore warrants checking tissue preservation, primary-antibody use, retrieval, and detection with controls (general IHC practice); it does not alone establish absent CGN. |
💡Expected CGN appearanceCall positive a cell-associated, predominantly apical junctional or membranous signal in the specified epithelium, potentially with cytoplasmic staining, and strong signal in HPA High cells; widespread nuclear or cell-independent haze is suspect (UniProt Q9P2M7; HPA tissue IHC; general IHC practice).
| Compartment and topology | CGN is on the cytoplasmic face of tight junctions and has no transmembrane segment (UniProt Q9P2M7). HPA also reports cytoplasmic and membranous tissue staining (HPA tissue IHC). Score the cell-border pattern with that resolution limit in mind; membrane staining alone does not prove precise tight-junction placement. |
| Choice of positive and comparison cells | HPA rates appendix, colon, and duodenal enterocytes, bronchial respiratory epithelium, and kidney collecting ducts High (HPA tissue IHC). Compare the named cell populations, because the HPA calls are cell-specific; a negative call for liver cholangiocytes does not negate UniProt’s broader report of liver expression (HPA tissue IHC; UniProt Q9P2M7). |
| Strength of antibody evidence | Four listed HPA antibodies have Enhanced IHC validation, while HPA reports medium consistency between staining and RNA expression (HPA antibodies; HPA tissue IHC). A matching distribution supports interpretation, but ambiguous staining still needs slide controls and cellular context (general IHC practice). |
| Isoforms and target modifications | UniProt lists two isoforms and multiple modified residues, including phosphoserines (UniProt Q9P2M7). The supplied record gives no antibody epitope or evidence that these features alter IHC staining; do not infer isoform-specific staining or a retrieval requirement from them. |
| Situation | Likely cause | Next action |
|---|---|---|
| Known-positive epithelium is blank. | The run may have a reagent, retrieval, or detection problem (general IHC practice); colon enterocytes are reported High (HPA tissue IHC). | Check a positive control in the same run, then verify primary-antibody application and detection steps (general IHC practice). The supplied sources specify no CGN-specific retrieval condition. |
| The slide shows mostly nuclear signal. | That location conflicts with junctional CGN and supported HPA plasma-membrane/cell-junction localization (UniProt Q9P2M7; HPA subcellular). | Check the no-primary control and examine whether expected epithelial borders stain (general IHC practice); avoid scoring isolated nuclei as CGN-positive. |
| Many unrelated cells stain strongly. | Cross-reactivity or endogenous detection activity is possible (general IHC practice); HPA staining levels are assigned to specific cell populations (HPA tissue IHC). | Review no-primary and detection controls, then compare each stained population with its HPA cell-level call (general IHC practice; HPA tissue IHC). |
| Background obscures cell boundaries. | Nonspecific binding or background from detection may be contributing (general IHC practice). | Use appropriate blocking and a no-primary control; adjust staining conditions only against a positive control so interpretable epithelial signal is retained (general IHC practice). |
| A nominally negative tissue contains focal staining. | The negative call may concern a different cell population: HPA reports liver cholangiocytes as Not detected, while UniProt reports liver expression (HPA tissue IHC; UniProt Q9P2M7). | Identify the stained cells before calling the result discordant; assess their compartment and control staining (general IHC practice). |
| Can ICC-IF help assess localization? | HPA supports plasma-membrane and cell-junction localization and lists ICC-IF images for A-431, U-251MG, and U2OS (HPA subcellular). | Use those observations as localization context, while interpreting the paraffin IHC slide against its tissue and cell-level evidence (HPA subcellular; HPA tissue IHC). |
Experiment evidence
IHC & IF images showing CGN staining
A02373 · IHCImmunohistochemistry of paraffin-embedded Human colon carcinoma using CGN Rabbit pAb at dilution of 1:100 .Perform microwave antigen retriev
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000143375-CGN/tissue/cerebral+cortex#imid_7236120
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000143375-CGN/tissue/colon#imid_7236062
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000143375-CGN/tissue/liver#imid_7236063
HPA · PancreasHuman Protein Atlas IHC, Pancreas. Source: https://www.proteinatlas.org/ENSG00000143375-CGN/tissue/pancreas#imid_7236006
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA027586; green = CGN, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA027586; green = CGN, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for CGN IHC & IF
🧪Run colon first: enterocytes are scored High for CGN, and staining should concentrate at their cell junctions (HPA: High in colon enterocytes; UniProt Q9P2M7: tight junction localization). Use heart muscle as a negative tissue, scoring cardiomyocytes, which are Not detected; on the colon slide, cells outside the enterocyte junctions should lack the specific junctional pattern, though nonspecific background may remain (HPA: Not detected in cardiomyocytes; UniProt Q9P2M7: tight junction localization).
Positive control tissue: Appendix (Enterocytes - Microvilli, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show CGN in A-431, U-251MG, U2OS, with annotated localisation: Plasma membrane (supported), Cell Junctions (supported) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control and a concentration-matched, nonimmune rabbit IgG isotype control for the rabbit polyclonal catalog antibody; confirm specificity with CGN knockout material or a peptide block when the immunizing peptide is available (selected A02373 caption: Rabbit pAb; standard IHC practice). In colon sections, quench endogenous peroxidase for chromogenic detection and check tissue autofluorescence if assessing IF (standard IHC/IF practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported in the supplied evidence; the selected A02373 paraffin-section caption also does not report a fixative (selected A02373 caption). That caption uses microwave retrieval in 10 mM PBS, pH 7.2, before staining at 1:100, but it does not establish that retrieval is required for every specimen (selected A02373 caption). IF can help resolve junctional localization, which HPA supports in ICC-IF images, but the evidence does not show that IF or frozen sections are easier than paraffin IHC; assess nonspecific colon background with the controls above (HPA: supported cell-junction localization; standard IHC/IF practice).
HPA tissue IHC evidence for CGN
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 | Enterocytes - Microvilli | High | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | High | Protein (IHC) | HPA → |
| Colon | Enterocytes - Microvilli | High | Protein (IHC) | HPA → |
| Duodenum | Enterocytes - Microvilli | High | Protein (IHC) | HPA → |
| Fallopian tube | Glandular cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Adrenal gland | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Cerebral cortex | Endothelial cells | Not detected | Protein (IHC) | HPA → |
| Heart muscle | Cardiomyocytes | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced CGN IHC Tips
CGN staining is most useful when junctional signal, tissue context, and processing controls agree (UniProt Q9P2M7; HPA tissue IHC).
Which retrieval condition should I try first for CGN in paraffin sections?
Start with citrate buffer at pH 6.0, heated to 95–98 °C for 20 min (page retrieval setting). If junctional staining is weak, compare adjacent sections using microwave retrieval in 10 mM PBS at pH 7.2, the condition shown for catalog antibody A02373 in paraffin-embedded human colon carcinoma (A02373 image caption). Keep antibody concentration and detection conditions constant across that comparison so retrieval is the variable being assessed (standard IHC practice). Score staining at epithelial cell contacts against background in the same sections; CGN is associated with tight junctions on the cytoplasmic face (UniProt Q9P2M7).
Could fixation explain weak CGN staining in my paraffin sections?
Target-specific fixation sensitivity is unknown from the supplied evidence; the A02373 paraffin-section caption does not state a fixative (A02373 image caption). Record the fixative, fixation duration, and processing history for each specimen before comparing staining intensity (standard IHC practice). If archived samples differ in processing, stain them in the same run with identical retrieval, antibody incubation, and chromogenic detection conditions (standard IHC practice). Include a known positive epithelial section and a no-primary control to distinguish a weak specimen from a run-wide failure or nonspecific color (HPA tissue IHC; standard IHC practice). Do not assign a fixation effect to CGN without a controlled comparison (standard IHC practice).
Where should convincing CGN staining appear in tissue architecture?
Look for staining concentrated along cell-cell contacts near the apical junctional region of polarized epithelia (UniProt Q9P2M7). CGN occupies the cytoplasmic face of tight junctions and has no annotated transmembrane segment, so a continuous extracellular surface pattern is unexpected (UniProt Q9P2M7). Compare the chromogenic pattern with tissue morphology and a matched positive section, such as colon epithelium, while keeping counterstain light enough to resolve cell borders (HPA tissue IHC; standard IHC practice). HPA reports cytoplasmic and membranous staining across several tissues, with abundant gastrointestinal staining; diffuse cytoplasmic color alone therefore needs junctional context and control review before interpretation (HPA tissue IHC).
Can CGN isoforms or epitope placement change how I interpret staining?
CGN has 2 annotated isoforms, but the supplied evidence does not map the catalog antibody epitope to either one (UniProt Q9P2M7; A02373 image caption). Consequently, a staining difference between specimens cannot be assigned to isoform abundance from this IHC assay alone (standard IHC interpretation). CGN has annotated phosphorylation sites, including serines 95 and 96, but their effect on this antibody's recognition is unreported (UniProt Q9P2M7; A02373 image caption). If epitope dependence matters, obtain the antibody immunogen or epitope information and compare with an independently validated reagent or orthogonal assay before making an isoform-specific claim (standard IHC practice).
How should I check a CGN junctional pattern by multiplex immunofluorescence?
Use IF/ICC as a separate validation workflow, pairing CGN with a marker that identifies the expected epithelial cell population in the specimen (UniProt Q9P2M7; standard IF practice). Choose fluorophores after inspecting unstained tissue for autofluorescence, and place the weaker target signal in the cleaner detection channel (standard IF practice). Because CGN is on the cytoplasmic face of tight junctions and lacks a transmembrane segment, test permeabilisation that permits intracellular antibody access (UniProt Q9P2M7; standard IF practice). Include single-label and no-primary controls to assess channel bleed-through and background, then compare junctional placement with the chromogenic IHC pattern without treating IF conditions as an IHC protocol (standard IF/IHC practice).
What should I adjust when CGN chromogenic staining looks diffuse or dirty?
First inspect a no-primary section and compare background with the putative junctional signal at epithelial contacts (UniProt Q9P2M7; standard IHC practice). For peroxidase-based chromogenic detection, verify the endogenous peroxidase block and shorten chromogen development if broad brown deposits obscure cell borders (standard IHC practice). Check that washing and protein blocking are consistent across sections, and titrate the primary antibody against a positive section; the A02373 image used 1:100 under its stated conditions (A02373 image caption; standard IHC practice). If background remains high, assess damaged tissue edges and necrotic areas separately from intact epithelium rather than scoring their color as CGN (standard IHC interpretation).
How should I score CGN IHC without losing junctional information? ⚠ ANSWER MARKED FOR VERIFICATION
Define a junctional compartment on intact epithelium before scoring, because CGN is associated with the apical junction complex (UniProt Q9P2M7). Report the percentage of epithelial cells with discernible junctional staining and an intensity-based H-score, recording diffuse cytoplasmic staining separately (standard IHC scoring practice; HPA tissue IHC). Normalize counts to the number of evaluable epithelial cells or junction length within the assessed region, and use the same region-selection rule across specimens (standard image-analysis practice). Exclude necrosis and section edges, document the chromogen threshold using controls, and avoid comparing raw stained area across specimens with different epithelial content (standard IHC scoring practice).
How can I distinguish a true CGN-positive result from artefact?
A credible result follows epithelial cell contacts near the apical junction and agrees with specimen morphology (UniProt Q9P2M7). HPA reports high staining in colon enterocyte microvilli and kidney collecting ducts, providing tissue context for a positive control, while its tissue profile also includes cytoplasmic and membranous expression (HPA tissue IHC). Treat isolated nuclear color, staining confined to damaged edges or necrosis, and widespread signal in the no-primary control as reasons to investigate artefact (UniProt Q9P2M7; standard IHC practice). For peroxidase detection, repeat the endogenous enzyme block or detection controls when brown deposits persist without primary antibody before assigning those deposits to CGN (standard IHC practice).
Boster reagents
Best CGN / Cingulin IHC Antibodies
A02373 has an IHC image from human paraffin-embedded colon carcinoma (A02373 image caption); A02373-1 lists human, mouse and rat IHC reactivity (catalog applications/reactivity). Neither lists IF validation (catalog applications/images).
Real IHC data
Anti-Cingulin CGN Antibody
Cat # A02373
The rendered A02373 card shows IHC on paraffin-embedded human colon carcinoma at 1:100 after microwave retrieval in 10 mM PBS, pH 7.2 (A02373 image caption). A02373-1 lists IHC for human, mouse and rat, but has no IHC image in the supplied catalog (A02373-1 applications/reactivity/images).
Which to pick: For tissue IHC, choose A02373 when the documented human paraffin-section example fits your sample; its caption does not report the fixative (A02373 image caption). For mouse or rat tissue, consider the rabbit polyclonal A02373-1, which lists IHC and reactivity with both species but supplies no processing example (A02373-1 catalog host, applications, reactivity and images). Neither SKU is listed for IF/ICC, so there is no catalog-supported IF/ICC pick here (catalog applications/images).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q9P2M7 (CING_HUMAN, Cingulin).
- Human Protein Atlas. CGN tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. CGN subcellular location (ICC-IF): Localized to the plasma membrane and cell junctions..
- Human Protein Atlas. CGN antibody validation summary (4 antibodies).
- The Roles and Regulatory Mechanisms of Tight Junction Protein Cingulin and Transcription Factor Forkhead Box Protein O1 in Human Lung Adenocarcinoma A549 Cells and Normal Lung Epithelial Cells. International journal of molecular sciences 2024 — PMC10855320.
- Histochemical study of cingulin and CD34 in the skin of a stray Dog. Open veterinary journal 2026 — PMC13314070.
- Inhibition of HDAC and Signal Transduction Pathways Induces Tight Junctions and Promotes Differentiation in p63-Positive Salivary Duct Adenocarcinoma. Cancers 2022 — PMC9179926.
- Exploring the Mechanism of Action of Danggui Buxue Decoction in Treating Chronic Glomerulonephritis Based on UPLC-MS/MS and Network Pharmacology. Immunity, inflammation and disease 2026 — PMC13564246.
- PubMed PMID:11042084 — UniProt-cited evidence.
- PubMed PMID:10718198 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for CGN (Cingulin). Expected localisation Cytoplasmic and membranous staining (HPA tissue IHC); apical junction expected (UniProt). Antigen retrieval: Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen). Validated IHC antibody: A02373 at 1:50-1:200 (the product's own IHC validation image is shown). Controls (HPA): positive Appendix (Enterocytes - Microvilli); negative Adipose tissue. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Paraffin-embedded Human colon carcinoma tissue; fixative not specified (datasheet A02373); 2. Fixation: Image fixative and duration unreported (datasheet A02373); 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-CGN, 1:50-1:200 (datasheet A02373); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep formalin fixation consistent across sections. (standard IHC practice; not target-specific); Caveat: Antibody staining and RNA show medium consistency (HPA tissue IHC); Isoform / epitope: 2 isoforms; check epitope coverage (UniProt); Regulation: Intestine-enhanced RNA expression (HPA tissue RNA). Evidence: UniProt Q9P2M7, HPA tissue IHC · Enhanced, HPA ICC-IF · plasma membrane and cell junctions., Datasheet A02373, PMC10855320, PMC13314070. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/CGN/immunohistochemistry-ihc-guide.