Background

Tight junctions (TJ) are multiprotein complexes localized at the apical-most portion of the intercellular junctional complexes that connect epithelial cells (Van Itallie and Anderson, 2014). These structures generate a permeability barrier and ion-specific paracellular pathways, maintain apico-basal polarity, provide input for various vital functions and modulate signaling pathways. The proteins located at the TJs can be divided into transmembrane and associated cytoplasmic proteins (reviewed in (González-Mariscal et al., 2003). The transmembrane proteins can be further subdivided into three major categories: claudins, tight junction-associated Marvel proteins (e.g., occludin) and single span proteins (e.g., Junctional Adhesion Molecules). TJ-associated cytosolic proteins include a large array of adaptors connected to signaling and cytoskeletal proteins. Collectively, these proteins are referred to as the cytoplasmic plaque. Claudins are small molecular weight (20-34 kDa) tetraspan membrane proteins that are essential components of the TJs (Tsukita et al., 2019). They incorporate into the TJ strands that typically contain a mosaic of various claudin isoforms that determine permselectivity (Van Itallie and Anderson, 2004). Interestingly, claudins have also recently emerged as key modulators of signaling, an effect that is likely due to the interactions with cytosolic adapters.

In this protocol we focus on claudin-2 (Cldn-2), a 230 amino acid member of the family, with a calculated molecular mass of 24.5 kDa. Cldn-2 was first described by Dr Shoichiro Tsukita and coworkers (Furuse et al., 1998a and 1998b) (for review see Venugopal et al., 2019). It is highly enriched in the kidney proximal tubules (Enck et al., 2001) and in intestinal and liver cells (e.g., Sakaguchi et al., 2002; Escaffit et al., 2005). It generates a cation selective paracellular channel, and therefore, its presence corresponds to elevated paracellular permeability (e.g., Amasheh et al., 2002). Multiple studies have revealed that in various cells Cldn-2 can be found not only at the membrane, i.e., in the TJs, but also in intracellular vesicles (Dukes et al., 2012; Lu et al., 2014; Amoozadeh et al., 2015), and in the nucleus (Ikari et al., 2014; Amoozadeh et al., 2018).

Many studies have used immunofluorescent staining in fixed cells followed by confocal microscopy to visualize TJ proteins and analyze their localization and abundance. These approaches allow investigation of the mechanisms that regulate TJ protein trafficking. The protocols differ in the fixation and blocking method and use primary antibodies targeted against specific proteins. Here we describe the method used in our lab to stain Cldn-2 in cultured kidney tubular cells based on (Amoozadeh et al., 2015 and 2018; Dan et al., 2019). We also detail our protocol for 3D reconstitution and analysis of changes in staining intensity following treatment, e.g. with cytokines. While we focus specifically on claudin-2, the same approach can be used for other TJ proteins in any epithelial or endothelial cells.