Alexa fluor 594 affinipure donkey anti guinea pig igg h l

A Click-iT EdU Alexa Fluor 488 Imaging Kit (Thermo Fisher Scientific, Germering, Germany) was used to perform 5-Ethynyl-2′deoxyuridine (Edu) labeling. Briefly, NPICCs were cultured in B-IC medium in 6-well plates for six days in the presence of butyrate (1000 µM). EdU (10 µM) was added to the medium during the last 72 h. Then islets were embedded in Histogel and paraffin, sectioned and used for EdU staining according to the manufacturer’s instructions. Tissues were permeabilized with 0.5% Triton X-100 for 20 min and incubated with the Click-iT reaction cocktail for 30 min at room temperature. Slices were stained with polyclonal guinea pig anti-insulin antibody (diluted 1:300, Agilent-Dako, Frankfurt, Germany) for 1 h, and secondary antibody Alexa Fluor^® 594 AffiniPure donkey anti-guinea pig IgG (H+L) for 45 min (1:1000, Jackson ImmunoResearch, Cambridgeshire, UK). Images were taken and quantified by Leica DM2500 microscope (Leica, Wetzlar, Germany) and Image J software (ImageJ 1.52a, http://rsb.info.nih.gov/ij/download.html, accessed on 23 April 2018). At least 1000 cells per each NPICC preparations were analyzed to count Edu labelled cells (n = 6).