LC3B Antibody Kit for Autophagy

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	1:100 dilution of Primary antibody

Protocol tips
1:100 dilution of Primary antibody

Publication protocol

The cells were fixed with 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA, USA) for 15 min, permeabilized with 0.1% Triton-X-100 (Sigma-Aldrich) for 15 min and blocked with Protein Block Serum-Free (Dako, Glostrup Municipality, Denmark) for 10 min. For centrosome staining, cells were fixed in ice-cold methanol for 10 minutes, rehydrated in PBS for 5 min, extracted in PBS-0.5% Triton X-100 for 5 min, blocked in TBS-BSA for 30 min, and then incubated with primary antibodies. The following primary antibodies were diluted in 1% BSA/PBS (Sigma-Aldrich) and added to the coverslips: anti-DCX, (1:300; Santa Cruz Biotechnology, Dallas, TX, USA), anti-Nestin (1:200; Millipore, Billerica, MA, USA), anti-Sox2 (1:500; Cell Signaling Technology, Danvers, MA, USA), anti-Ki67 (1:1000; ThermoFisher Scientific), anti-cleaved Caspase-3 (1:400; Cell Signaling Technology), anti-LC3B (1:100; ThermoFisher Scientific); anti-alpha-tubulin (1:1000; Sigma-Aldrich) and anti-pericentrin (1:1000, Abcam, Cambridge, UK). For ZIKV staining, we used a primary monoclonal antibody produced against Flavivirus E protein (MIAF, obtained through the WRECVA, diluted 1:2000), kindly provided by Dr. Nikos Vasilakis (University of Texas, Medical Branch). Alternatively, a serum sample obtained from a local subject with confirmed ZIKV infection in the past 6 months, was used for ZIKV staining, 1:500 dilution (Fig. 6). All the antibodies were incubated overnight at 4 °C. The following secondary antibodies were used, in 1:1000 dilution: anti-mouse IgG Alexa Fluor 488, anti-rabbit IgG Alexa Fluor 568, anti-goat IgG Alexa Fluor 488, anti-mouse IgG Alexa Fluor 488, anti-human IgG Alexa Fluor 488 (all from ThermoFisher Scientific). Nuclei staining was performed with DAPI (Vector Laboratories, Burlingame, CA, USA). Images captured on A1+ confocal microscope (Nikon, Tokyo, Japan) or FluoView 1000 confocal microscope (Olympus, Tokyo, Japan). Quantifications were performed in seven random fields captured under 200x magnification, using the Image Pro Plus v.7.0 software (Media Cybernetics, Rockville, MD, USA.

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