For confocal microscopy, three 40 μm-thick skin paraffin-embedded specimens from HS-NL and healthy AGR sample groups were assessed, as previously described [10 (link)]. Imaging of the immunostained samples was carried out on an Olympus FV3000 confocal system using a 40× or 60× oil-immersion lens (NAs: 1.40–1.42, respectively). Acquisition settings (laser power, confocal aperture and gain, detector parameters) were identical for all samples. Series of 1 um thick optical sections with 0.5 µm separation in the Z axis were acquired. The overall number of optical sections for each sample was 6. No pixels corresponding to immunostained puncta were saturated. Images for the figures were processed using the Adobe Photoshop CS5 software.
Regularity of the CLDN and DSG1 immunostaining was analyzed by measuring the distance between two adjacent immunostained puncta along the cross section of the cell membrane (defined as a closed line around a DAPI stained nucleus). Inter-puncta distances (120–160 per condition) were measured in the samples. The distances were presented as box plots where the box range represents +/− SD, whiskers indicate the 1.5-fold IQR distance (Q3–Q1) outliers. All datapoints are plotted as grey dots. The mean value is shown as a hollow square within the box.
Regularity of the CLDN and DSG1 immunostaining was analyzed by measuring the distance between two adjacent immunostained puncta along the cross section of the cell membrane (defined as a closed line around a DAPI stained nucleus). Inter-puncta distances (120–160 per condition) were measured in the samples. The distances were presented as box plots where the box range represents +/− SD, whiskers indicate the 1.5-fold IQR distance (Q3–Q1) outliers. All datapoints are plotted as grey dots. The mean value is shown as a hollow square within the box.
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