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CLDN2 / Claudin-2 · IHC design guide
Design Immunohistochemistry for CLDN2
Plan CLDN2 paraffin-section IHC around membranous staining in gallbladder, kidney and GI (HPA tissue IHC). This guide covers tissue controls, fixation, detection and interpretation, with a starting antibody concentration of 0.5–1 μg/mL (datasheet: RP1042).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
CLDN2 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 | Membranous staining in gallbladder, kidney and GI (HPA tissue IHC) | |
| Staining pattern | Membranous glandular and proximal tubule staining (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen) | |
| Positive control | Gallbladder+1 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 | Presumed off-target binding may confound staining (HPA tissue IHC) | |
| Regulation | Expression regulation not annotated (UniProt) | |
| Isoform / epitope | No isoforms; extracellular and cytoplasmic epitopes differ (UniProt) |
Section 1
Recommended CLDN2 IHC & IF Protocols
Compare the catalog antibody’s IHC-P protocol with published CLDN2 protocols for goat jejunum (PMC4847856), human lung tissue microarrays (PMC10366158), and pancreatic FFPE samples (PMC13222116).
| Sample | Tissue sections; selected-image fixative not specified (standard IHC workflow) |
| Fixation | Image fixative and duration unreported (datasheet RP1042); 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-CLDN2, 0.5-1μg/ml (datasheet RP1042) |
| 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 | CLDN2-positive staining in glandular cells of gallbladder (HPA tissue IHC: High). HPA tissue profile: Membranous expression in gall bladder, kidney and GI. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6.0 HIER at 95–98 °C for 20 min (page retrieval rule); the goat protocol reports EDTA pretreatment (PMC4847856).
Section 2
What Is the Expected CLDN2 Staining Pattern?
CLDN2 is a 4-pass membrane protein at tight junctions (UniProt P57739 topology; UniProt P57739 subcellular). In paraffin-section IHC, expect membranous staining in gallbladder glandular cells and kidney proximal tubules (HPA: gallbladder High; kidney Medium). HPA rates its tissue IHC profile Enhanced, while noting medium agreement with RNA data and presumed off-target staining that was disregarded (HPA: tissue IHC reliability).
| Distinct staining outlines gallbladder glandular cells, with less intense staining in kidney proximal tubules. | This fits HPA's membranous profile and its High gallbladder versus Medium kidney levels (HPA: tissue IHC). Judge the relevant cells and their borders; a whole-section average can obscure the expected cell pattern. |
| Strong nuclear or broadly cytoplasmic chromogen dominates the expected epithelial borders. | That is discordant with the main tight-junction and membrane localization (UniProt P57739 subcellular; HPA: tissue IHC). Treat it as possible nonspecific staining until controls support it; HPA's additional nuclear and vesicular locations come from ICC-IF, not tissue IHC (HPA: subcellular). |
| The same conspicuous signal appears in the expected cells and in unrelated cells, including cells in a comparison section. | Consider cross-reactivity or endogenous detection activity before scoring those extra cells as CLDN2. HPA reports presumed off-target tissue staining that it disregarded (HPA: tissue IHC reliability). Appendix glandular cells are a useful comparison because HPA reports them as not detected (HPA: appendix). |
| A diffuse tint covers tissue, lumina, or the slide rather than tracing cell borders. | This cannot establish the membranous pattern reported by HPA (HPA: tissue IHC). Review the negative detection control and background across the section; in routine IHC, excess reagent signal or incomplete washing can obscure localization. |
| Gallbladder glandular cells lack detectable staining in a section expected to be positive. | The result conflicts with HPA's High gallbladder finding (HPA: gallbladder). Check the assay and section before interpreting absence as biology. A negative result from one preparation does not by itself overturn a tissue-level reference profile. |
💡Expected CLDN2 appearanceCall a section positive when staining is predominantly membranous in gallbladder glandular cells or kidney proximal tubules, with the stronger reference level in gallbladder (High) than kidney (Medium); prominent diffuse or wrong-cell staining is suspect (HPA: tissue IHC; UniProt P57739 subcellular).
| Membrane topology | CLDN2 has 4 transmembrane segments, extracellular loops, and cytoplasmic regions (UniProt P57739 topology). Interpret a border-associated pattern in light of this location; topology alone does not specify an antibody's epitope or an antigen-retrieval condition. |
| Cell and tissue distribution | HPA reports High gallbladder glandular staining, Medium kidney proximal-tubule staining, and Low staining in stomach, duodenum, small intestine, and seminal-vesicle glandular cells (HPA: tissue IHC). Match the named cell type when choosing comparisons. |
| Tissue IHC confidence | HPA calls the tissue profile Enhanced but describes medium agreement between antibody staining and RNA expression; presumed off-target staining was disregarded (HPA: tissue IHC reliability). Use localization and comparison tissue together when judging an unexpected signal. |
| Antibody-specific validation | HPA lists CAB002609 as IHC Enhanced; its other listed antibodies have no supplied IHC rating (HPA: antibodies). That rating does not establish the performance of a different catalog antibody, so evaluate that antibody against the expected tissue pattern. |
| Processing and fixation evidence | UniProt lists one chain spanning residues 1–230, with no signal peptide, propeptide, or annotated glycosylation site (UniProt P57739 processing). These features do not establish fixation sensitivity; no target-specific fixation effect is supplied. |
| Situation | Likely cause | Next action |
|---|---|---|
| Gallbladder glandular cells are blank. | A technical failure is possible because HPA reports High staining in those cells (HPA: gallbladder). The supplied sources do not identify a CLDN2-specific retrieval failure. | Confirm the expected cells are present; review antibody dilution, detection reagents, and the run's positive control. If optimizing retrieval, document it as general IHC optimization rather than a known CLDN2 requirement. |
| Only diffuse cytoplasmic or nuclear stain is visible. | The compartment conflicts with the main membrane and junction location (UniProt P57739 subcellular; HPA: tissue IHC). ICC-IF additional locations do not validate dominant nuclear tissue-IHC staining (HPA: subcellular). | Compare cell borders with the counterstain and a negative detection control; adjust general background controls before assigning a CLDN2-positive score. |
| Appendix glandular cells stain as strongly as gallbladder glandular cells. | HPA reports appendix glandular cells as not detected and gallbladder glandular cells as High (HPA: tissue IHC). Cross-reactivity or endogenous detection activity may explain the discrepancy. | Inspect negative detection controls and localization in both sections; record the discordance. An HPA 'not detected' observation is a reference pattern, not proof that every appendix specimen must be blank. |
| Low-level GI staining looks negative beside gallbladder. | HPA rates stomach, duodenum, and small-intestine glandular cells Low, versus High gallbladder glandular cells (HPA: tissue IHC). Direct intensity comparison can hide a weak localized result. | Score the named glandular cells and membrane pattern in each tissue separately. Keep exposure or chromogen development comparable within the run when assessing relative intensity. |
| Background obscures the epithelial borders. | Diffuse signal prevents assessment of the membranous tissue profile (HPA: tissue IHC); nonspecific reagent binding or endogenous detection activity are routine IHC possibilities. | Check negative detection controls, blocking and washing, then adjust antibody or detection conditions according to the assay. Do not infer a CLDN2-specific fixation effect from background alone. |
| Can an ICC-IF nuclear or vesicular signal be used to score tissue IHC? | HPA's supported main ICC-IF locations are plasma membrane and cell junctions; nucleoplasm, nucleoli, and vesicles are additional approved locations (HPA: subcellular). | Use the tissue-IHC membranous profile to score paraffin sections (HPA: tissue IHC). Evaluate ICC-IF localization separately; it does not supply an IHC protocol or validate nuclear chromogen staining. |
Experiment evidence
IHC & IF images showing CLDN2 staining
RP1042 · IHCAnti-Claudin 2 antibody, RP1042, IHC(P) IHC(P): Human Intestinal Cancer Tissue
A03033-1 · IFIF analysis of Claudin 2/CLDN2 using anti-Claudin 2/CLDN2 antibody (A03033-1). Claudin 2/CLDN2 was detected in an immunocytochemical section
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000165376-CLDN2/tissue/cerebral+cortex#imid_810992
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000165376-CLDN2/tissue/colon#imid_810910
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000165376-CLDN2/tissue/liver#imid_810919
HPA · GallbladderHuman Protein Atlas IHC, Gallbladder. Source: https://www.proteinatlas.org/ENSG00000165376-CLDN2/tissue/gallbladder#imid_810887
HPA · ICC-IF A-549Human Protein Atlas immunofluorescence, A-549, antibody HPA051548; green = CLDN2, blue = nucleus, red = microtubules.
HPA · ICC-IF A-549Human Protein Atlas immunofluorescence, A-549, antibody HPA051548; green = CLDN2, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for CLDN2 IHC & IF
🧪Run gallbladder first: its glandular cells should show junctional membrane staining (HPA: High in gallbladder glandular cells; UniProt P57739: tight junction localization). Use adipose tissue as the negative tissue (HPA: Not detected in adipocytes); on the gallbladder slide, adjacent nonglandular cells should lack specific junctional staining, but treat them as a background check because their CLDN2 status is not reported (HPA: gallbladder glandular cells only).
Positive control tissue: Gallbladder (Glandular cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show CLDN2 in A-549, CACO-2, U2OS, RPTEC/TERT1, hTERT-RPE1 (serum starved), with annotated localisation: Plasma membrane (supported), Cell Junctions (supported) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control and an isotype control matched to the primary antibody’s host species and clonality; use CLDN2 knockout tissue or peptide competition, if available, as a biological specificity control (standard IHC practice). For chromogenic staining of gallbladder, quench endogenous peroxidase and check residual background (standard IHC practice).
⚠️Feasibility: A target-specific fixation window, fixation effect, and antigen-retrieval dependency are unreported in the supplied evidence; optimize retrieval for paraffin IHC (standard IHC practice). The RP1042 IHC(P) caption reports human intestinal cancer tissue but no fixative, so the fixative is unreported (caption: RP1042). The supplied evidence does not establish whether frozen sections or IF are easier, and reports no gallbladder-specific artefact (supplied target/application evidence).
HPA tissue IHC evidence for CLDN2
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — Medium consistency between antibody staining and RNA expression data. Presumed off target binding observed and disregarded.). 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 |
|---|---|---|---|---|
| Gallbladder | Glandular cells | High | Protein (IHC) | HPA → |
| Kidney | Proximal tubules (cell body) | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Appendix | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | Not detected | Protein (IHC) | HPA → |
| Caudate | Glial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced CLDN2 IHC Tips
Troubleshoot CLDN2 staining in paraffin sections by checking retrieval, tissue preservation, junctional localisation and cell-specific scoring.
What retrieval should I try when CLDN2 junctional staining is weak?
Start with citrate pH 6.0 HIER at 95–98 °C for 20 min (page IHC retrieval rule). CLDN2 spans the membrane 4 times, so assess whether staining outlines cell junctions rather than merely increasing overall colour (UniProt P57739 topology and localisation). Compare adjacent sections with identical antibody dilution, detection and counterstain; include a gallbladder glandular-cell control (HPA: High in gallbladder glandular cells). If signal remains weak, test EDTA pH 9.0 as a fallback and compare tissue integrity and junctional contrast (standard IHC practice). Treat diffuse staining that appears only after harsher retrieval cautiously (UniProt P57739 localisation).
Could fixation explain variable CLDN2 staining between paraffin blocks?
Target-specific fixation sensitivity is unknown: the selected tissue-IHC caption reports paraffin sections but does not state a fixative (RP1042 IHC(P) caption). Record each block’s fixative and fixation time before attributing a staining difference to CLDN2 expression (standard IHC practice). Where matched material is available, compare sections processed under documented conditions, keeping retrieval, antibody dilution and chromogen development identical (standard IHC practice). Check morphology and junctional contrast together, since damaged epithelium can make membrane scoring unreliable (standard IHC practice; UniProt P57739 localisation). Do not infer fixation tolerance from HPA staining patterns or CLDN2 topology (HPA tissue IHC; UniProt P57739 topology).
How can I distinguish junctional CLDN2 staining from nonspecific cytoplasmic colour?
Prioritise crisp staining along epithelial cell contacts, consistent with CLDN2’s tight-junction and membrane localisation (UniProt P57739 localisation). Compare that pattern with a positive gallbladder glandular-cell section and a negative-control section processed in the same run (HPA: High in gallbladder glandular cells; standard IHC practice). A diffuse cytoplasmic blush without contact enrichment warrants review of retrieval, antibody concentration, wash stringency and chromogen development (standard IHC practice). HPA also reports nucleoplasmic, nucleolar and vesicular signals in subcellular assays; those observations alone do not establish that nuclear or vesicular colour in a paraffin section is specific (HPA subcellular). Score compartments separately when patterns disagree (standard IHC practice).
How should I interpret discordant results from CLDN2 antibodies with different epitopes?
Check each antibody’s documented immunogen or epitope before comparing stains; the supplied record does not identify the catalog antibody’s epitope (record; standard IHC practice). CLDN2 has extracellular regions at residues 29–81 and 138–162, plus a cytoplasmic tail at 184–230 (UniProt P57739 topology). Phosphoserines at 219 and 223 may matter for an antibody directed to that tail, but epitope dependence has not been established here (UniProt P57739 modified residues). No isoforms are annotated, so investigate retrieval, epitope accessibility and compartment-specific background before assigning discordance to a splice form (UniProt P57739 isoforms; standard IHC practice).
How can IF help resolve an ambiguous chromogenic CLDN2 pattern?
Use IF on a matched section to examine whether CLDN2 follows epithelial cell contacts, while retaining chromogenic IHC as the primary readout (UniProt P57739 localisation; standard IF practice). Multiplex with an epithelial marker such as pan-cytokeratin, and consider ZO-1 to assess junction alignment (standard IF practice; UniProt P57739 subunit). Select a far-red fluorophore if green tissue autofluorescence obscures signal, and include single-stain and unstained controls (standard IF practice). If the antibody recognises the cytoplasmic tail at 184–230, test mild permeabilisation; an extracellular epitope may be assessed without it (UniProt P57739 topology; standard IF practice). Confirm epitope location before interpreting that comparison (standard IF practice).
What should I change when CLDN2 IHC shows widespread background?
First inspect no-primary and secondary-only controls to separate antibody-associated colour from detection background (standard IHC practice). Block endogenous peroxidase before chromogenic development, then compare controlled reductions in primary concentration or incubation time without changing retrieval simultaneously (standard IHC practice). Assess nonspecific edge staining, precipitate and staining over damaged tissue separately from continuous epithelial junctions (standard IHC practice; UniProt P57739 localisation). HPA reports presumed off-target binding in its tissue assessment, so a broadly positive section requires compartment and cell-type checks rather than acceptance based on colour alone (HPA tissue IHC reliability). Recheck wash steps and DAB development against the same-run positive control (standard IHC practice).
How should I quantify CLDN2 across heterogeneous paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define epithelial regions before scoring and record the percentage of cells with convincing junctional staining (UniProt P57739 localisation; standard IHC practice). For an H-score, grade membrane intensity 0–3, multiply each grade by its percentage of cells and sum to obtain 0–300 (standard IHC practice). Normalise positive-cell counts to evaluable epithelial cells, or membrane-positive length to evaluable epithelial boundary length, rather than total section area (standard IHC practice). Exclude folds, necrosis and missing epithelium using predefined rules, and report compartments separately if cytoplasmic colour is present (standard IHC practice). Keep scoring thresholds and imaging conditions fixed across the comparison (standard IHC practice).
When is a CLDN2-positive IHC result credible?
A credible result shows epithelial junctional staining in the expected compartment and reproduces in the same-run control (UniProt P57739 localisation; standard IHC practice). HPA reports high staining in gallbladder glandular cells and medium staining in kidney proximal tubules, providing contextual controls rather than universal expression rules (HPA tissue IHC). Examine unexpected nuclear colour, staining in an unrelated cell population, section-edge enhancement and necrotic areas as possible artefacts (HPA subcellular; standard IHC practice). Use a no-primary control to assess endogenous enzyme or detection colour, and compare intact neighbouring tissue before calling a focal signal positive (standard IHC practice). Interpret the selected intestinal-cancer image cautiously because its fixative is unreported (RP1042 IHC(P) caption).
Boster reagents
Best CLDN2 / Claudin-2 IHC Antibodies
Anti-CLDN2 antibodies have image data for paraffin-section IHC in human intestinal cancer tissue (RP1042 IHC image caption) and IF/ICC in human Caco-2 cells (A03033-1 IF image caption).
Real IHC data
Anti-Claudin 2/CLDN2 Antibody ®
Cat # RP1042
Real IF data
Anti-Claudin 2/CLDN2 Antibody ®
Cat # A03033-1
RP1042 is listed for IHC and shows staining in a human intestinal cancer paraffin section (RP1042 applications; RP1042 IHC image caption). A03033-1 is listed for IF/ICC and shows staining in human Caco-2 cells (A03033-1 applications; A03033-1 IF image caption).
Which to pick: Choose RP1042 for tissue IHC: its image shows a paraffin section, its human IHC-P dilution is 0.5–1 μg/mL, and the fixative is unreported (RP1042 IHC image caption; RP1042 datasheet). Choose A03033-1 for IF/ICC in human cells; its pictured Caco-2 assay used 5 μg/mL (A03033-1 applications; A03033-1 IF image caption). For rat work, RP1042 is the only SKU listing rat reactivity, although its pictured IHC sample is human; clonality is unreported for both antibodies (catalog reactivity; RP1042 IHC image caption; catalog clone fields).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P57739 (CLD2_HUMAN, Claudin-2).
- Human Protein Atlas. CLDN2 tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. CLDN2 subcellular location (ICC-IF): Mainly localized to the plasma membrane and cell junctions. In addition localized to the nucleoplasm, nucleoli and vesicles..
- Human Protein Atlas. CLDN2 antibody validation summary (3 antibodies).
- Expression of Tight Junction Proteins and Cadherin 17 in the Small Intestine of Young Goats Offered a Reduced N and/or Ca Diet. PloS one 2016 — PMC4847856.
- Multidirectional characterization of cellular composition and spatial architecture in human multiple primary lung cancers. Cell death & disease 2023 — PMC10366158.
- Claudin-2 protects against colitis-associated cancer by promoting colitis-associated mucosal healing. The Journal of clinical investigation 2023 — PMC10688979.
- Inflammation-Induced Claudin-2 Up-Regulation Limits Pancreatitis Development by Enhancing Pancreatic Ductal Transport. Gastroenterology 2026 — PMC13222116.
- PubMed PMID:11934881 — UniProt-cited evidence.
- PubMed PMID:12975309 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for CLDN2 (Claudin-2). Expected localisation Membranous staining in gallbladder, kidney and GI (HPA tissue IHC). Antigen retrieval: Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen). Validated IHC antibody: RP1042 at 0.5-1μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Gallbladder (Glandular cells); negative Adipose tissue. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Tissue sections; selected-image fixative not specified (standard IHC workflow); 2. Fixation: Image fixative and duration unreported (datasheet RP1042); 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-CLDN2, 0.5-1μg/ml (datasheet RP1042); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across sections. (standard IHC practice; not target-specific); Caveat: Presumed off-target binding may confound staining (HPA tissue IHC); Isoform / epitope: No isoforms; extracellular and cytoplasmic epitopes differ (UniProt); Regulation: Expression regulation not annotated (UniProt). Evidence: UniProt P57739, HPA tissue IHC · Enhanced, HPA ICC-IF · Mainly localized to the plasma membrane and cell junctions. In…, Datasheet RP1042, PMC4847856, PMC10366158. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/CLDN2/immunohistochemistry-ihc-guide.