TEWL was determined by directly measuring the water evaporation from the dorsal skin of newborn mice using a moisture analyzer NEP-1 BRAVO (MECCO Inc.).
TJ permeability assay using surface biotinylation technique was performed according to the method developed by Chen et al. (1997) (link). 50 μl of 10 mg/ml EZ-Link™ Sulfo-NHS-LC-Biotin (Pierce Chemical Co.) in PBS containing 1 mM CaCl2 was injected into the dermis on the back of the Cln1+/+ and Cln1−/− newborns. After 30-min incubation, the skin was taken out and frozen in liquid nitrogen. About 5-μm-thick frozen sections were fixed in 95% ethanol at 4°C for 30 min and then in 100% acetone at room temperature for 1 min. The sections were soaked in blocking solution for 15 min, incubated with antioccludin mAb for 30 min, washed three times with blocking solution, then incubated with a mixture of FITC anti–rat IgG pAb (Jackson ImmunoResearch Laboratories) and Streptavidin Texas red (Oncogene Research Products) for 30 min.
TJ permeability assay using surface biotinylation technique was performed according to the method developed by Chen et al. (1997) (link). 50 μl of 10 mg/ml EZ-Link™ Sulfo-NHS-LC-Biotin (Pierce Chemical Co.) in PBS containing 1 mM CaCl2 was injected into the dermis on the back of the Cln1+/+ and Cln1−/− newborns. After 30-min incubation, the skin was taken out and frozen in liquid nitrogen. About 5-μm-thick frozen sections were fixed in 95% ethanol at 4°C for 30 min and then in 100% acetone at room temperature for 1 min. The sections were soaked in blocking solution for 15 min, incubated with antioccludin mAb for 30 min, washed three times with blocking solution, then incubated with a mixture of FITC anti–rat IgG pAb (Jackson ImmunoResearch Laboratories) and Streptavidin Texas red (Oncogene Research Products) for 30 min.
