Occludin-labels are associated with TJ and non-TJ structures.

<p>(A) A projection image of deeper layers of the same area of the epidermal sheet shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031641#pone-0031641-g001" target="_blank">Figure 1D</a>. (A′-A″″) 3D images of the third (3), fourth (4) and fifth (5) cells from the uppermost nucleated cell layers of the square in A. (B, C) Occludin immunoelectron microscopy. The labels are associated with TJs (brackets) in SG1 and SG2, but with small dots on the cell membrane in the lower epidermis (C″) (arrow). Silver intensified nanogold labels (B) and 5 nm-sized gold labels (C). B′, C′ and C″ are high-magnification views of B and C. Bars = 10 µm (A), 500 nm (B, C) and 200 nm (B′, C′, and C″).</p

Secreted LGs are frequently found in the lanthanum-positive spaces.

<p>Lower (A, B) and higher (C) magnification views of a lanthanum penetration assay performed on normal human skin. LGs secreted into the lanthanum-positive extracellular spaces are outlined in red and those in the cytoplasm are outlined in green. In the 5 µm-width (blue arrows) area of a SG2 cell, secreted LG-areas are 0.510 µm² and cytoplasmic ones are 0.376 µm². The point where lanthanum stops is indicated with a yellow arrow. Lanthanum is lost during tissue preparation in the dilated intercellular spaces in the layers below SG3.</p

LGs start being secreted before establishment of paracellular permeability barrier.

<p>(A, B) TJ permeability assay using a surface biotinylation technique. LG molecules, CDSN and glucosylceramides (GlcCer) are co-localized with biotin (areas between arrowheads). A part of CDSN/biotin image in A is magnified in A′ and the points where the tracer stops are indicated with yellow arrows. The basement membrane zone is highlighted by the broken line. (C, D) Lanthanum penetration assay on normal human skin. (C) Lanthanum penetration is blocked at the TJ (bracket) in the SG1. (D) Secreted LGs (red arrows) can be found in the tracer permeable intercellular space. Green arrows indicate LGs in the cytoplasm. The area in the rectangle with broken lines in D is magnified in D′. D, desmosome. Bars = 10 µm (A, A′, B), 200 nm (C, D′) and 500 nm (D).</p

CDSN is found in the intercellular space (ICS) where a biotin tracer is detected.

<p>This figure is the result of the paracellular permeability assay using a biotin tracer performed on normal human skin. The areas in the rectangles in panel A are magnified in panels B and C. CDNS labels (red arrows) are seen in the biotin tracer negative ICS between SC and SG1 associating with corneodesmosomes (CD) (B), but they are in the tracer (blue arrows) positive ICS between SG1 and SG2 (C). Larger dots are silver intensified streptavidin/gold particles reacted with biotin. Smaller dots are 10 nm gold particles labelling CDSN. CDSN labels associated with LGs in the cytoplasm are indicated with green arrows. D, desmosome. Bars = 1 µm (A) and 200 nm (B, C).</p

Multilayered TJ-protein immunoreactive networks in the human epidermis.

<p>(A, A′) Biotin-tracer passed through some occludin-positive dots (arrows), but stopped at the most superficial dots (arrowhead). (B, B′) The CDSN-positive SG1 and SG2 have occludin-positive dots (arrows). (C, D) Occludin immunostaining of a sweat duct (C) and an epidermal sheet (D). D and D″ show a projection image of the upper three nucleated cell layers. D″ shows a 3D image from the corner (arrow). D′″ shows slice views dissecting uppermost (1), the second (2) and the third (3) cells. (E, F) EM of TJs (brackets) in the sweat duct (E) and the epidermis (F). A′, B′ and D′ are high-magnification views of A, B and D respectively. Bars = 10 µm (A–D′) and 200 nm (E, F).</p