A11004 alexa fluor 568

HaCaT cells or 1° KC were grown on 10 mm² coverslips at a density of 70’000 cells/coverslip in 24 wells plates. Cells were fixed in 4% paraformaldehyde (PFA) in PBS for 10 min at RT, permeabilized with 0.25% of Triton X-100 in PBS for 10 min and then blocked in PBS containing 1% BSA at room temperature for 30 min as previously described [44 (link)]. Coverslips were incubated with primary antibody ASC (Cat. NBP1-78977 Novus Biological, Littleton, CO, USA) 1:100, NLRP1 (sc-166368 Santa Cruz Biotechnology Inc., Dallas, TX, USA) 1:50, 4HNE (ab46545, Abcam, Cambridge, UK) 1:400, DPP9 (ab42080, Abcam, Cambridge, UK)1:400, UBR2 (18852-1-AP, Proteintech Group Inc., Rosemont, IL, USA) 1: 150 in 0.25% BSA/PBS overnight at 4°C. The Alexa Fluor Fluorochrome-conjugated secondary antibodies (A11004 Alexa Fluor 568, A11008 Alexa Fluor 488 Invitrogen, ThermoFisher Scientific, USA) were used at a dilution of 1:1000 in PBS-BSA 0.25% for 1 h at RT. Nuclei were stained with DAPI (D1306 Invitrogen ThermoFisher Scientific, Waltham, MA, USA) after removal of secondary antibody. Coverslips were mounted onto glass slides using PermaFluor Aqueous Mounting Medium (TA-006-FM, ThermoFisher Scientific, Waltham, MA, USA), and examined using a Zeiss Z1 AxioObserver LSM10 confocal microscope equipped at 40x and 60x magnification. Images were quantified using ImageJ software.