Alexafluor488 anti guinea pig

Human pancreatic islets of non-diabetic subjects were cultured with standard medium, or in the presence/absence of IGFBP3 and/or in the presence/absence of Ecto-TMEM219 for 72 h as previously described (see “Pancreatic islets and Interventional studies”). Islets were detached with Versene (ThermoFisher Scientific) and stained with the BD Horizon™ Fixable Viability Stain 510 (FVS510, BD Biosciences 564406, San Jose, CA), which ensures accurate assessment of cell viability in samples after fixation and/or permeabilization as per manufacturer’s instructions. After being stained with BD Horizon™ Fixable Viability Stain 510, cells were next fixed and permeabilized with Fixation and Permeabilization Solution Kit (554714, BD Biosciences, San Jose, CA)^{51 (link)} and finally stained with guinea pig anti-insulin antibody (1:200, Guinea Pig, ThermoFisher Scientific, PA1-26938) followed by AlexaFluor488 anti-guinea pig (1:200, ThermoFisher Scientific, A-11073). Flow cytometry analysis was performed using a BD FACS Celesta flow cytometry system (BD Biosciences) and analyzed using Flowjo software (Version 6 and Version 10, Tree Star, Ashland, OR).

Polytene chromosome preparation, FISH analysis and indirect immunostaining were performed according to the protocol described in [38 (link)].
Rabbit polyclonal anti-Fibrillarin (Abcam, ab5821, 1:5000) or guinea pig anti-Udd (1:300, [46 (link)], kindly provided by M. Buszczak) primary antibodies and Alexa Fluor 488 anti-guinea pig or/and Alexa Fluor 568-conjugated goat anti-rabbit secondary antibodies (1:500; Thermo Fisher Scientific, Waltham, MA, USA) were used for immunostaining.