Biotinylated anti mouse

Double immunofluorescence was performed on deparaffined and permeabilized hMSG sections using rabbit anti-AQP5 (1:100; Millipore, Burlington, MA, USA), mouse anti-ezrin (1:100; Thermo-Fisher Scientific, Waltham, MA, USA), anti-rabbit antibody-conjugated Alexa 488 (1:1000; Cell Signaling, Danvers, MA, USA), and biotinylated anti-mouse (1:200; Jackson ImmunoResearch, West Grove, PA, USA) followed by a streptavidin-anti-mouse conjugated-Alexa594 (1:100; Sigma-Aldrich, St-Louis, MI, USA). Immunofluorescent labeling of ezrin was quantified on the images captured at 20× magnification using a Leica DM 2000 microscope. One microscopic field, generally containing the whole section, was analyzed for each sample. Tissue containing acini was selected and the reacting surfaces were quantified using CellSens Imaging Software (Olympus, Düsseldorf, Germany). The color threshold was calculated on negative controls. Image analysis was performed using the percentage of the reacting area and the level of pixel color intensity per field. The degree of ezrin reactivity was calculated as the product between the average of the positive area percentage and the mean value of pixel color intensity per microscopic field.

Histological preparations were obtained from leprosy patients’ skin. Tissue slides embedding was done through freezing using Tissue Tec OCT compound (Sakura) and liquid nitrogen. After sectioning, slides were fixed in 10% formalin. Endogenous phosphatase blocking was made using 5% acetic acid, and 2.5% goat serum was used for unspecific sites. Primary scFv pure antibody (265.8 μg/mL) incubation was done overnight. Secondary and tertiary antibodies, 1:200 anti-His (GE Healthcare) produced in mouse and 1:300 biotinylated anti-mouse (Jackson Lab, code 115.065.003) were used, respectively. Microscopic slides were incubated with the avidin-biotin alkaline-phosphatase complex (1:100, Jackson Lab) and development of the colorimetric reaction was done with fast red Naphthol. A counterstain was done using Harris’ Hematoxylin.