Claudin 3 (HMV309)

Claudin 3 (CLDN3) is a tight-junction (TJ) protein with four-transmembrane domains which is coded by the intron-less CLDN3 gene on chromosome 7q11.23. It is one of at least 27 members of the claudin protein family. Tight junctions (TJs) are cell-cell adhesion structures in epithelial and endothelial tissues that form a continuous seal around cells and act as a physical barrier to prevent (or enable) the passing of water and molecules through the paracellular space. Claudin-3 is a barrier forming and rather ubiquitously expressed TJ protein and also acts as a low-affinity receptor for Clostridium perfringens enterotoxin. Aberrant expression has been described to occur in cancer and was related to poor patient prognosis. Despite the widespread expression of CLDN3 in normal cells, it is considered a putative drug target, since the accessibility to TJ proteins is limited by the orchestrated cell growth in normal tissues, and the lower level of orientation may result in higher drug exposure of cancer cells. 

Images describing the Claudin 3 staining pattern in normal tissues obtained by the antibody HMV309 are shown in our “Normal Tissue Gallery”.

In normal tissues, CLDN3 is extensively expressed in many epithelial cell types, especially in the intestine, but also in many other tissues. These findings are largely consistent with the RNA data described in the Human Protein Atlas (Tissue expression Claudin 3). 

Positive control = Liver: A strong membranous CLDN3 staining should be seen in bile ducts. In addition, a weak to moderate membranous CLDN3 staining should be seen in hepatocytes (often seen predominantly at the bile secreting pole).

Negative control = Stomach: Most epithelial cells should be CLDN3 negative, while a strong membranous CLDN3 staining of a small subset of epithelial cells in the neck and in glandular pits must be seen.

Normal tissue gallery

Cancer tissue gallery

No data available at the moment

IHC users have different preferences on how the stains should look like. Some prefer high staining intensity of the target stain and even accept some background. Others favor absolute specificity and lighter target stains. Factors that invariably lead to more intense staining include higher concentration of the antibody and visualization tools, longer incubation time, higher temperature during incubation, higher temperature and longer duration of the heat induced epitope retrieval (slide pretreatment). The impact of the pH during slide pretreatment has variable effects and depends on the antibody and the target protein.

All images and data shown here and in our image galleries are obtained by the manual protocol described below. Other protocols resulting in equivalent staining are described as well.

Manual protocol

Freshly cut sections should be used (less than 10 days between cutting and staining). Heat-induced antigen retrieval for 5 minutes in an autoclave at 121°C in pH 7,8 Target Retrieval Solution buffer. Apply HMV309 at a dilution of 1:150 at 37°C for 60 minutes. Visualization of bound antibody by the EnVision Kit (Dako, Agilent) according to the manufacturer’s directions.

• The diagnostic and prognostic relevance of CLDN3 expression in tumors and in preneoplastic disease needs to be investigated.
• CLDN3 is a putative therapeutic target in cancer.
• The role of CLDN3 in non-neoplastic disease needs further investigation.

There are two ways how the specificity of antibodies can be documented for immunohistochemistry on formalin fixed tissues. These are: 1. Comparison with a second independent method for target expression measurement across a large number of different tissue types (orthogonal strategy), and 2. Comparison with one or several independent antibodies for the same target and showing that all positive staining results are also seen with other antibodies for the same target (independent antibody strategy). 

Orthogonal validation: For the antibody HMV309 specificity is consistent with the particularly strong immunostaining in tissue types, for which three independent RNA screening studies, including the Human Protein Atlas (HPA) RNA-seq tissue dataset, the FANTOM5 project, and the Genotype-Tissue Expression (GTEx) project, which are all summarized in the Human Protein Atlas (Tissue expression Claudin 3) had demonstrated the highest RNA levels such as the intestine, thyroid, pancreas, and the prostate. CLDN3 positivity by HMV309 was, however, also detectable in several tissues without documented CLDN3 RNA expression (germinal centre cells in lymphatic tissues, megakaryocytes in the bone marrow, squamous epithelium positivity in the thymus and the tonsil crypts, gallbladder, urothelium, placenta, epididymis, gastric mucosa, adrenal gland, and the parathyroidal gland) as well as in tissues with only very low CLDN3 RNA levels (endometrium) Some of these tissues without previously documented CLDN3 RNA expression but CLDN3 positivity by HMV309 had only very few positive cells that were probably not detected in RNA studies (megakaryocytes, urothelial cells, corpuscles of Hassall’s). For several other tissues, these discrepancies were more difficult to comprehend.

Comparison of antibodies: True expression of CLDN3 in all cell types found to be CLDN3 positive by HMV309 is corroborated by identical staining’s obtained by another commercially available independent antibody (termed “validation antibody”). This specifically applies to cell types from organs that were considered CLDN3 RNA negative (or only weakly positive) such as germinal centre cells in lymphatic tissues, megakaryocytes in the bone marrow, squamous epithelium in the thymus and the tonsil crypts, gallbladder, urothelium, endometrium, placenta, epididymis, gastric mucosa, adrenal gland, and the parathyroid gland. Because of the significant discrepancies with RNA data, the antibody validation also included a second independent commercial validation antibody which also confirmed all these stainings (and additionally showed a cross-reactivity with a gastric mucosa protein) (data not shown).

Normal tissue gallery