For histological evaluation of HND, a histological database at the Yonsei University Severance Hospital was utilized. A query search of AD patients from 2011 who underwent facial skin biopsy was performed, and five patients were randomly selected from 9 candidates.
Histological analysis of non-HND face specimens was performed through a query search of AD patients who underwent skin biopsy on the face from 2013 for suspected concomitant vitiligo (usually the biopsy is conducted with non-lesional normal skin and lesional skin with vitiligo to compare the melanocyte population). Crude age filtering was performed to age-match AD patients. Among the 10 candidates, five patients were randomly selected for image analysis.
At 200x magnification, the longest distance from the subcorneal level to the basal layer was chosen arbitrarily for epidermal thickness after calibrating the scale bar to pixels. The number of vessels/mm2 was counted in the dermis of each slide section within a 100 µm distance from the epidermal–dermal junction.
Immunohistochemical staining was performed using paraffin-embedded sections with antibodies against factor VIII-related antigen (1:100, ab236284, Abcam), stromal cell-derived factor-1-alpha (SDF1-α) (1:100, ab25117, Abcam, Cambridge, United Kingdom), Interleukin-1-beta (IL-1-β) (1:100, ab2105, Abcam), tumor necrosis factor-alpha (TNF-α) (1:50, ab1793, Abcam), transforming growth factor-beta (TGF-β) (1:100, ab66043, Abcam), and vascular endothelial growth factor (VEGF) (1:200, ab1316, Abcam). Staining intensity was determined at 400x magnification at a randomly chosen area of the upper dermis. Images were quantified using ImageJ analysis tools (National Institutes of Health, Bethesda, MA).
To calculate the stained area of the antibody, we converted the original image to an 8-bit grayscale image (ImageJ>Image>8-bit), applied a binary threshold, and calculated the percentage positive for the stained part in the standard image. Quantification was performed relative to the entire selected region. The threshold for each staining was set as the average threshold of multiple immunostaining analyses performed by three independent experimenters.
Histological analysis of non-HND face specimens was performed through a query search of AD patients who underwent skin biopsy on the face from 2013 for suspected concomitant vitiligo (usually the biopsy is conducted with non-lesional normal skin and lesional skin with vitiligo to compare the melanocyte population). Crude age filtering was performed to age-match AD patients. Among the 10 candidates, five patients were randomly selected for image analysis.
At 200x magnification, the longest distance from the subcorneal level to the basal layer was chosen arbitrarily for epidermal thickness after calibrating the scale bar to pixels. The number of vessels/mm2 was counted in the dermis of each slide section within a 100 µm distance from the epidermal–dermal junction.
Immunohistochemical staining was performed using paraffin-embedded sections with antibodies against factor VIII-related antigen (1:100, ab236284, Abcam), stromal cell-derived factor-1-alpha (SDF1-α) (1:100, ab25117, Abcam, Cambridge, United Kingdom), Interleukin-1-beta (IL-1-β) (1:100, ab2105, Abcam), tumor necrosis factor-alpha (TNF-α) (1:50, ab1793, Abcam), transforming growth factor-beta (TGF-β) (1:100, ab66043, Abcam), and vascular endothelial growth factor (VEGF) (1:200, ab1316, Abcam). Staining intensity was determined at 400x magnification at a randomly chosen area of the upper dermis. Images were quantified using ImageJ analysis tools (National Institutes of Health, Bethesda, MA).
To calculate the stained area of the antibody, we converted the original image to an 8-bit grayscale image (ImageJ>Image>8-bit), applied a binary threshold, and calculated the percentage positive for the stained part in the standard image. Quantification was performed relative to the entire selected region. The threshold for each staining was set as the average threshold of multiple immunostaining analyses performed by three independent experimenters.
Free full text: Click here
