Alexa 488 and alexa 647 conjugated secondary antibodies

For immunocytochemistry, cells grown on poly-L-lysine-coated coverslips were fixed with 4% paraformaldehyde for 10 min and permeabilized with 0.5% Triton X-100 in PBS for 10 min. After washing step with PBS, cells were incubated in a blocking solution (2% goat serum in PBS) and then incubated with primary antibodies. For immunohistochemistry, mouse brain sections were prepared according to the method previously described24 (link). The following primary antibodies were used: anti-p62 (CST), anti-ubiquitin (Santa Cruz), anti-αS (CST), anti-CHOP (Santa Cruz), anti-phospho-neurofilament (Covance), anti-phospho-c-Jun (CST), and anti-cleaved caspase-3 (CST). Positive immunostaining was detected using Alexa 488- and Alexa 647-conjugated secondary antibodies (Molecular Probes). Nuclei were counterstained with DRAQ7 (BioStatus) or TO-PRO3 iodide (Molecular Probes). Fluorescence images were analyzed with an FV300 confocal laser scanning microscope (Olympus).

Horseradish peroxidase-conjugated secondary anti-IgG antibodies were purchased from Millipore (Billerica, MA, USA). Alexa 488- and Alexa 647-conjugated secondary antibodies were obtained from Molecular Probes (Eugene, OR, USA). Polyclonal rabbit anti-HSV-1 antibody was from Dako. Living Colors anti-reef coral fluorescent protein (RCFP) polyclonal pan antibody was from Clontech. Anti-LAMP-1 mouse monoclonal antibody H4A3 was from DSHB (Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, Iowa, USA). Sheep anti-TGN46 polyclonal antibody was from Serotec. Low-glucose Dulbecco modified Eagle (DMEM), fetal bovine serum (FBS), pooled human serum, carboxymethylcellulose sodium salt (CMC), and medium-viscosity and protease inhibitor cocktail were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Mowiol was from Calbiochem (Merck Chemicals, Germany).