Keratinocytes were fixed with 3% paraformaldehyde for 10 minutes. When nuclei staining was required, cells were incubated with DAPI (4′,6-diamidino-2-phenylindole) for 15 min and cells were mounted in glass slides using Mowiol. Images from necrosis and autophagy assays were acquired in Olympus
Provis BX51 microscope coupled to monochromatic camera SPOT RT using SimplePCI 6 software (Hamamatsu, Japan). For cell morphology, phase contrast images were acquired with 10X objective and for video time-lapse acquisition, real time phase contrast images were acquired every 5 min, during 6 hours with 40X objective in
Widefield Zeiss Axio Observer microscope, using Zen acquisition software (Carl Zeiss AG, Oberkochen, Germany). For LAAO internalization analysis, LAAO and Bovine Serum Albumin (BSA) labeling was performed using
Alexa Fluor® 555 Microscale Protein Labeling Kit (ThermoFisher Scientific, Leicestershire, United Kingdom) as per manufacturer’s instructions. Images were acquired using 60X objective in
Widefield Zeiss Axio Observer microscope, using Zen acquisition software (Carl Zeiss AG, Oberkochen, Germany).
Costal-Oliveira F., Stransky S., Guerra-Duarte C., Naves de Souza D.L., Vivas-Ruiz D.E., Yarlequé A., Sanchez E.F., Chávez-Olórtegui C, & Braga V.M. (2019). L-amino acid oxidase from Bothrops atrox snake venom triggers autophagy, apoptosis and necrosis in normal human keratinocytes. Scientific Reports, 9, 781.
