Mouse anti ezrin

Proximity ligation assays (PLAs) were performed using Duolink kit (Sigma-Aldrich, St-Louis, MI, USA) according to the manufacturer’s instructions. PLAs were performed on paraffin-embedded hMSG sections using rabbit anti-AQP5 (1:100; Millipore, Burlington, MA, USA) and mouse anti-ezrin (1:100; Thermo-Fisher Scientific, Waltham, MA, USA). PLAs were performed on methanol-fixed transfected NS-SV-AC cells using mouse anti-HA-tag (1:100; Proteintech, Rosemont, IL, USA) and rabbit anti-ezrin (1:200; Cell Signaling, Danvers, MA, USA). Negative controls were performed in the absence of one or both antibodies. Z-stack images were acquired using a confocal microscope (LSM-710) with an ×63/1.4 PlanApochromat lens (Zeiss, Oberkochen, Germany) and processed as previously described [12 (link)].

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.