Cells stained with pSTAT-1 antibodies were classified as macrophages M1 and those with CD163, CD206, and CMAF staining were classified as M2.7,11The cells were quantified in duplicate, and the results were expressed as number of stained cells per square millimeter. The quantification method was the same as that used in a previous study conducted by our group.4 The number of cells immunostained with specific antibodies in the superficial and middle dermis (portion of the epidermis where the pathophysiological events of urticaria occur) was enumerated at a magnification of 400× in 12 optical fields, using an optical microscope (Zeiss™, Axiophot, Germany). The density of immunostained cells was calculated with the following equation:
Cell density/mm2 = (immunostained cells in 12 fields)/12/0.0625.
The variability of the duplicate count was not greater than 5%.
Blank immunohistochemistry stains were performed, where the primary antibodies were excluded, which were negative.