CLDN18 / Claudin-18 · IHC design guide

Design Immunohistochemistry for CLDN18

Plan chromogenic CLDN18 IHC in paraffin sections around the membranous staining reported in stomach glands and alveolar cells (HPA tissue IHC). This guide covers fixation consistency, antibody use and interpretation of the expected epithelial pattern.

Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use

Immunohistochemistry protocol sheet for CLDN18 (IHC for CLDN18): expected localisation Observed epithelial membranes; tight junctions expected (HPA tissue IHC; UniProt), antibody A09129-1, validated IHC image, and IHC protocol steps — Printable **CLDN18** IHC protocol sheet — expected localisation Observed epithelial membranes; tight junctions expected (HPA tissue IHC; UniProt), antibody A09129-1, controls and protocol steps. Open the full CLDN18 IHC guide →

Navigate to: Protocols Staining pattern Controls Antibodies

CLDN18 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	Observed epithelial membranes; tight junctions expected (HPA tissue IHC; UniProt)
	Staining pattern	Membranous in stomach glands, gallbladder epithelium and alveoli (HPA tissue IHC)
	Antigen retrieval	EDTA pH 8.0 HIER, heat-mediated (datasheet A09129-1)
	Positive control ⓘ	Stomach+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 tissue fixation consistent. (standard IHC practice; not target-specific)
	Caveat	Gallbladder glandular staining is low (HPA tissue IHC)
	Regulation	No expression regulator established (UniProt; HPA tissue IHC)
	Isoform / epitope	A1 and A2; epitope side may matter, but maps are unspecified (UniProt)

Section 1

Recommended CLDN18 IHC & IF Protocols

Start with the catalog antibody’s IHC-P protocol (datasheet A09129-1), then compare the three published CLDN18 IHC protocols below (PMC12103281; PMC11271439; PMC12975664).

Recommended immunohistochemistry (IHC-P) protocol parameters
Sample	Paraffin-embedded human gastric cancer tissue; fixative not specified (datasheet A09129-1)
Fixation	Image fixative and duration unreported (datasheet A09129-1); 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 A09129-1); 20 min, 95–100 °C (standard)
Peroxidase block	3% H2O2, 10 min, room temperature (standard)
Blocking	10% goat serum (datasheet A09129-1)
Primary antibody	Rabbit anti-CLDN18, 2-5 μg/ml (datasheet A09129-1)
Primary incubation	Overnight at 4 °C (datasheet A09129-1)
Detection	HRP-conjugated secondary, DAB chromogen (datasheet A09129-1)
Counterstain	Hematoxylin, blue, dehydrate and mount (standard)
Expected result	CLDN18-positive staining in glandular cells of stomach (HPA tissue IHC: High). HPA tissue profile: Distinct membranous expression in stomach, gallbladder epithelium and alveolar cells. No signal in the no-primary control.

💡Decision noteTry heat-mediated EDTA, pH 8.0, retrieval first for the catalog antibody (datasheet A09129-1); the automated TMA protocol specifies CC1 retrieval (PMC11271439).

Section 2

What Is the Expected CLDN18 Staining Pattern?

CLDN18 is a four-pass membrane protein localized to epithelial tight junctions and lateral membranes (UniProt P56856 topology and subcellular location). In paraffin-section IHC, expect membranous staining in stomach glandular cells, lung alveolar type II cells, and gallbladder epithelium (HPA tissue IHC). HPA rates the tissue staining profile Enhanced, citing high consistency between antibody staining and RNA expression data (HPA tissue IHC).

What am I looking at on my slide?
Distinct membrane outlines in stomach glandular cells; weaker outlines in alveolar type II cells (HPA tissue IHC).	This fits the reported High stomach and Medium lung staining levels and the expected junctional or lateral membrane location (HPA tissue IHC; UniProt P56856 subcellular location). Compare staining within the relevant cell type rather than expecting uniform intensity across tissues.
Predominantly nuclear staining, or diffuse cytoplasmic staining without discernible membrane outlines.	This does not match the annotated tight-junction and lateral membrane location (UniProt P56856 subcellular location). Treat it as a localization discrepancy and investigate background or nonspecific binding before scoring it as CLDN18 positivity (general IHC practice).
Strong signal in adipocytes or bronchial respiratory epithelial cells.	HPA reports CLDN18 as Not detected in those cell types (HPA tissue IHC). Cross-reactivity or endogenous chromogen-generating activity are possible explanations, but the slide alone cannot distinguish them; review detection controls (general IHC practice).
Color spread across cells, stroma, and empty areas, with poorly defined cell borders.	Diffuse background obscures the membranous pattern reported by HPA (HPA tissue IHC). Review reagent-only and primary-omission controls, blocking, and washing before interpreting faint tissue signal (general IHC practice).
No membrane signal in stomach glandular cells on a run intended to detect CLDN18.	Stomach glandular cells are a reported High-staining reference (HPA tissue IHC). A blank reference calls for a run-level check of antibody, detection reagents, retrieval, and tissue integrity; it does not by itself establish that the test tissue lacks CLDN18 (general IHC practice).

💡Expected CLDN18 appearanceCall a result positive when epithelial cell borders show distinct membranous staining, strongest in stomach glandular cells and moderate in alveolar type II cells (HPA tissue IHC; UniProt P56856 subcellular location); isolated nuclear color or diffuse haze is not the expected pattern (UniProt P56856 subcellular location; general IHC practice).

How each factor affects the staining
Membrane topology and epitope location (UniProt P56856 topology).	CLDN18 has four transmembrane segments, two extracellular loops, and cytoplasmic regions (UniProt P56856 topology). Epitope location could affect antibody access, but no epitope assignment or target-specific retrieval effect is supplied; use the chosen antibody's documented IHC conditions (general IHC practice).
Tissue and cell-type distribution (HPA tissue IHC; UniProt P56856 tissue specificity).	HPA reports High stomach glandular, Medium alveolar type II, and Low gallbladder glandular staining (HPA tissue IHC). UniProt describes expression as restricted to lung (UniProt P56856 tissue specificity). Keep that source difference visible when choosing reference tissue; do not assume their distribution statements agree.
Antibody validation (HPA antibody records; HPA tissue IHC).	HPA marks HPA018446 and CAB013243 Enhanced for IHC, and CAB013010 Supported (HPA antibody records). Its overall tissue profile is Enhanced (HPA tissue IHC). These ratings support comparison with the reported pattern; they do not establish the performance of an unspecified catalog antibody.
Isoforms and processing (UniProt P56856).	UniProt lists A1 and A2 isoforms, no signal peptide or propeptide, and a 1–261 chain (UniProt P56856). The supplied record gives no antibody epitope or isoform coverage, so a negative stain cannot identify which isoform is absent. No shedding-based interpretation is supported.
IF/ICC Q: What location should a separate IF/ICC assessment check?	A: Cell junctions; HPA calls this location Approved for ICC-IF (HPA subcellular). Its images include OE19, U-251MG, and U2OS (HPA subcellular). Those image listings do not establish paraffin-section staining intensity or an IF/ICC protocol.

Why is my staining missing, weak or wrong?
Situation	Likely cause	Next action
Stomach reference is blank while the detection control works.	Antibody conditions, antigen retrieval, or tissue quality may need review (general IHC practice); stomach glandular staining is reported High (HPA tissue IHC).	Check the antibody's documented IHC-P conditions, retrieval setup, reagent preparation, and section integrity (general IHC practice). No CLDN18-specific fixation sensitivity is established by the supplied sources.
Lung signal seems weaker than stomach signal.	The difference may match HPA's Medium alveolar type II versus High stomach glandular levels (HPA tissue IHC).	Compare the correct cells and membrane outlines against the stomach reference before changing conditions (HPA tissue IHC; general IHC practice). Avoid calling lung negative solely because its signal is less intense.
Signal appears mainly nuclear or as broad cytoplasmic fill.	That compartment conflicts with the reported junctional and lateral membrane location (UniProt P56856 subcellular location).	Review the primary-omission control, counterstain, and antibody conditions; score only convincing membrane-localized signal (general IHC practice; UniProt P56856 subcellular location).
Unexpected strong color appears in HPA-negative cell types.	Cross-reactivity or endogenous detection activity may contribute (general IHC practice); HPA reports adipocytes and bronchial respiratory epithelial cells as Not detected (HPA tissue IHC).	Compare primary-omission and reagent controls, confirm cell identity, and reassess antibody specificity before assigning CLDN18 positivity (general IHC practice).
Background obscures epithelial borders throughout the section.	Nonspecific binding, inadequate blocking or washing, or detection background can obscure a membrane pattern (general IHC practice).	Inspect control sections and optimize blocking, washes, and the documented antibody conditions until membrane borders can be evaluated (general IHC practice).
Gallbladder has faint staining despite a clear stomach reference.	HPA describes gallbladder glandular staining as Low while its profile notes membranous expression in gallbladder epithelium (HPA tissue IHC).	Assess whether faint signal follows epithelial membranes and whether background controls are clean (HPA tissue IHC; general IHC practice). Do not require stomach-level intensity in gallbladder.

Source: UniProt P56856 Human Protein Atlas tissue Human Protein Atlas subcellular

Experiment evidence

IHC & IF images showing CLDN18 staining

IHC analysis of Claudin18/CLDN18 using anti-Claudin18/CLDN18 antibody (A09129-1). Claudin18/CLDN18 was detected in a paraffin-embedded section of human gastric cancer tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2 μg/ml rabbit anti-Claudin18/CLDN18 Antibody (A09129-1) overnight at 4°C. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using HRP Conjugated Rabbit IgG Super Vision Assay Kit (Catalog # SV0002) with DAB as the chromogen.

A09129-1 · IHCIHC analysis of Claudin18/CLDN18 using anti-Claudin18/CLDN18 antibody (A09129-1). Claudin18/CLDN18 was detected in a paraffin-embedded secti

IF analysis of Claudin18/CLDN18 using anti-Claudin18/CLDN18 antibody (A09129-1). Claudin18/CLDN18 was detected in an immunocytochemical section of Hela cells. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent (AR0022) for 15 mins. The cells were blocked with 10% goat serum. And then incubated with 5 μg/mL rabbit anti-Claudin18/CLDN18 Antibody (A09129-1) overnight at 4°C. DyLight®488 Conjugated Goat Anti-Rabbit IgG (BA1127) was used as secondary antibody at 1:100 dilution and incubated for 30 minutes at 37°C. The section was counterstained with DAPI. Visualize using a fluorescence microscope and filter sets appropriate for the label used.

A09129-1 · IFIF analysis of Claudin18/CLDN18 using anti-Claudin18/CLDN18 antibody (A09129-1). Claudin18/CLDN18 was detected in an immunocytochemical sect

HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000066405-CLDN18/tissue/cerebral+cortex#imid_3156116

HPA · StomachHuman Protein Atlas IHC, Stomach. Source: https://www.proteinatlas.org/ENSG00000066405-CLDN18/tissue/stomach+2#imid_3155992

HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000066405-CLDN18/tissue/colon#imid_3156050

HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000066405-CLDN18/tissue/liver#imid_3156035

HPA · ICC-IF OE19Human Protein Atlas immunofluorescence, OE19, antibody HPA018446; green = CLDN18, blue = nucleus, red = microtubules.

← swipe / scroll for more images →

Real IHC / IF data behind the staining-pattern expectations in this section.

Sample controls for CLDN18 IHC & IF

🧪Run stomach first: glandular cells should show high CLDN18 staining (HPA: High in stomach glandular cells). Use adipose tissue adipocytes as a negative control (HPA: Not detected in adipocytes); on the stomach slide, compare glandular membrane staining with adjacent non-glandular cells as an internal background reference, confirming their staining status by morphology.

Positive control tissue: Stomach (Glandular cells, HPA High)

Negative control tissue: Adipose tissue (HPA Not detected)

ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show CLDN18 in OE19, U-251MG, U2OS, with annotated localisation: Cell Junctions (approved) (HPA subcellular).

Technical controls: Include a no-primary, secondary-only control and a concentration-matched rabbit isotype control (selected A09129-1 caption: rabbit primary antibody). Use CLDN18 knockout material as a biological negative where available, and quench endogenous peroxidase before chromogenic detection in stomach sections (standard IHC practice).

⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the exact A09129-1 paraffin-section caption does not state the fixative (selected A09129-1 caption). That caption uses heat retrieval in EDTA at pH 8.0, but does not establish that retrieval is required; it supports paraffin-section IHC without showing that frozen sections or IF are easier (selected A09129-1 caption). In stomach sections, assess glandular membranes separately from luminal material or nonspecific chromogen deposits when scoring the junctional pattern (HPA: High in stomach glandular cells; UniProt P56856: epithelial tight junction localization; standard IHC practice).

HPA tissue IHC evidence for CLDN18

Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — High 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
Stomach	Glandular cells	High	Protein (IHC)	HPA →
Lung	Alveolar cells type II	Medium	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 →
Appendix	Glandular cells	Not detected	Protein (IHC)	HPA →
Bone marrow	Hematopoietic cells	Not detected	Protein (IHC)	HPA →
Breast	Adipocytes	Not detected	Protein (IHC)	HPA →

Section 3

Advanced CLDN18 IHC Tips

For CLDN18 chromogenic IHC in paraffin sections, evaluate junctional staining alongside retrieval, tissue controls, and the antibody’s documented epitope specificity.

How should I optimize retrieval when CLDN18 staining is weak in paraffin sections?

Start with heat-mediated retrieval in EDTA pH 8.0 for paraffin sections (datasheet A09129-1). Keep section thickness, heating time, and cooling conditions consistent while comparing retrieval runs; record these variables so changes in junctional signal can be attributed to the retrieval step (standard IHC practice). Include a stomach glandular-cell control, where CLDN18 staining is high, and inspect whether staining remains membranous (HPA tissue IHC). If signal is weak, test a modest change in heating time on adjacent sections before considering another buffer as a fallback (standard IHC practice). Excess retrieval can damage morphology, making epithelial borders and scoring less reliable (standard IHC practice).

Could fixation explain weak or uneven CLDN18 staining?

The selected paraffin-section caption does not state a fixative, so target-specific fixation sensitivity cannot be determined from this evidence (datasheet A09129-1). Record the fixative, time to fixation, fixation duration, and section age for each specimen before comparing staining intensity (standard IHC practice). Process a known positive stomach control alongside the test sections; its glandular cells show high CLDN18 staining (HPA tissue IHC). If staining varies between specimens, compare similarly processed blocks and inspect tissue preservation before changing antibody concentration (standard IHC practice). Do not attribute a weak result specifically to CLDN18 epitope loss without a controlled fixation comparison (datasheet A09129-1; standard IHC practice).

Which staining pattern should count as a credible CLDN18 positive result?

Look for membrane-associated staining at epithelial cell contacts, consistent with CLDN18 localisation to tight junctions and the lateral membrane (UniProt P56856 localisation). In tissue, distinct membranous staining has been reported in stomach and gallbladder epithelium and alveolar cells (HPA tissue IHC). Compare the test section with stomach glandular cells, reported as high, and lung alveolar type II cells, reported as medium (HPA tissue IHC). Score convincing junctional or lateral membrane signal separately from diffuse cytoplasmic colour, which does not match the documented principal location (UniProt P56856 localisation; HPA tissue IHC). Check morphology at high magnification before calling faint cell borders positive (standard IHC practice).

Can this stain distinguish CLDN18 isoforms or extracellular from cytoplasmic epitopes?

CLDN18 has 2 listed isoforms, A1 and A2, but the supplied antibody caption does not establish isoform selectivity or give an epitope sequence (UniProt P56856 isoforms; datasheet A09129-1). The protein has 4 transmembrane segments, extracellular loops at residues 28–80 and 144–174, and a cytoplasmic tail at 196–261 (UniProt P56856 topology). Consequently, membranous staining alone cannot identify the bound loop, tail, or isoform (UniProt P56856 topology; standard IHC interpretation). Obtain epitope mapping or isoform-specific validation before assigning an A1 or A2 result (standard antibody-validation practice). Report staining as CLDN18 unless that additional evidence is available (standard antibody-validation practice).

How should I adapt CLDN18 localisation checks for a multiplex IF experiment?

For IF/ICC, pair CLDN18 with a marker that identifies the expected epithelial population, then assess whether CLDN18 outlines cell junctions within those cells (UniProt P56856 localisation; HPA tissue IHC; standard IF practice). Choose fluorophores after measuring tissue autofluorescence in an unstained control, and move the weaker signal into a cleaner spectral channel (standard IF practice). Match permeabilisation to the validated epitope: access to the cytoplasmic tail at 196–261 may require it, whereas an extracellular-loop epitope should first be tested without it (UniProt P56856 topology; standard IF practice). The supplied IHC caption provides no IF/ICC fixation or permeabilisation validation for this antibody (datasheet A09129-1). Confirm channel separation and junctional localisation with single-stain controls (standard IF practice).

What should I change when DAB background obscures CLDN18 cell borders?

First distinguish widespread deposit from the expected epithelial membrane pattern at cell contacts (UniProt P56856 localisation; HPA tissue IHC). The documented paraffin-section example used 10% goat serum blocking, 2 μg/ml primary antibody overnight at 4°C, a peroxidase-conjugated secondary for 30 minutes at 37°C, and DAB detection (datasheet A09129-1). Use a no-primary control to identify detection-system background, and include a peroxidase-blocking step for chromogenic IHC (standard IHC practice). If background persists, titrate primary antibody and DAB development while keeping the retrieval condition fixed (standard IHC practice). Evaluate any improvement against a positive tissue control so reduced background is not mistaken for improved sensitivity (HPA tissue IHC; standard IHC practice).

How should I score heterogeneous CLDN18 staining across a tissue section? ⚠ ANSWER MARKED FOR VERIFICATION

Define a scoring region before reviewing the stain, exclude folds and necrosis, and count interpretable epithelial cells within that region (standard IHC practice). Report the percentage of cells with convincing membrane staining and intensity by category; an H-score can summarize percentage-weighted intensity on a 0–300 scale (standard IHC scoring practice). Normalise positive-cell counts to the total eligible epithelial-cell count, or report stained junctional area per measured epithelial area when architecture permits (standard IHC practice). Keep cytoplasmic colour outside the primary membrane score because CLDN18 is documented at tight junctions and lateral membranes (UniProt P56856 localisation). Apply the same threshold and control section across cases (standard IHC practice).

How can I separate true CLDN18 staining from tissue or detection artefacts?

A credible result follows epithelial cell borders or junctions, matching CLDN18’s documented membrane and tight-junction localisation (UniProt P56856 localisation; HPA tissue IHC). Stomach glandular cells provide a high reference, whereas adipocytes and several other listed cell populations are reported as not detected (HPA tissue IHC). Treat isolated nuclear staining, diffuse colour in unrelated cells, and signal confined to section edges or necrotic areas as suspect pending control review (UniProt P56856 localisation; standard IHC practice). Check a no-primary section and the peroxidase block when brown deposit appears in unexpected compartments (standard chromogenic IHC practice). Interpret stomach staining with the tissue evidence in mind despite UniProt’s lung-restricted tissue statement (HPA tissue IHC; UniProt P56856 tissue specificity).

Boster reagents

Best CLDN18 / Claudin-18 IHC Antibodies

A09129-1 has real IHC images from paraffin-embedded human gastric cancer and rat stomach sections (catalog IHC captions), plus IF images from those sections and Hela cells (catalog IF captions).

Real IHC data

Anti-Claudin18/CLDN18 Antibody ®

Cat # A09129-1

View product →

A09129-1 is the sole card and lists human and rat reactivity with IHC, IF, and ICC applications (catalog: applications; reactivity). Its images document IHC in paraffin-embedded human gastric cancer and rat stomach sections and IF in those sections and Hela cells (catalog IHC/IF captions).

Which to pick: Choose A09129-1 for chromogenic tissue IHC: its own paraffin-section captions document EDTA retrieval at pH 8.0, 2 μg/ml primary antibody, and HRP/DAB detection (A09129-1 IHC captions). For IF/ICC, the same SKU lists both applications and shows IF in Hela cells and paraffin-embedded human gastric cancer and rat stomach sections at 5 μg/ml primary antibody (catalog: applications; A09129-1 IF captions). It covers human and rat samples (catalog: reactivity); clonality and tissue fixative are unreported.

Each figure is that product's own IHC / IF validation image from its datasheet.

References

UniProt Consortium. UniProt entry P56856 (CLD18_HUMAN, Claudin-18).
Human Protein Atlas. CLDN18 tissue IHC expression (reliability: Enhanced).
Human Protein Atlas. CLDN18 subcellular location (ICC-IF): Localized to the cell junctions..
Human Protein Atlas. CLDN18 antibody validation summary (3 antibodies).
Claudin 18.2 Expression in 1404 Digestive Tract Adenocarcinomas Including 1175 Colorectal Carcinomas: Distinct Colorectal Carcinoma Subtypes Are Claudin 18.2 Positive. Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc 2025 — PMC12103281.
Claudin-18.2 immunohistochemical evaluation in pancreatic cancer specimens: review and recommendations for routine testing and scoring. Virchows Archiv : an international journal of pathology 2025 — PMC12488767.
Spatial expression of claudin 18.2 in matched primaries and metastases of tubo-ovarian carcinoma of all subtypes. Virchows Archiv : an international journal of pathology 2024 — PMC11271439.
Claudin-18 expression in gastric type adenocarcinoma and HPV-associated adenocarcinoma of the uterine cervix. Histopathology 2026 — PMC12975664.
PubMed PMID:11585919 — UniProt-cited evidence.
PubMed PMID:12975309 — UniProt-cited evidence.
PubMed PMID:15489334 — UniProt-cited evidence.