Monoclonal anti synaptophysin

Hippocampal 450 μm-thick transversely coronal slices were treated or not with sCT PFOs- or Monomer-enriched solutions for 100 min. After treatment, the slices were fixed over night with 4% paraformaldehyde in PBS, 0.12 M in sucrose. After fixation, the samples were rinsed three times in PBS with 5% sucrose and 0.15 mM CaCl₂ and left overnight in sucrose buffer (PBS with 30% sucrose and 0.15 mM CaCl₂). Samples were then embedded in Tissue Freezing Medium (Jung, Germany), frozen at −30 °C in isopentane and stored at −80 °C. Sections (8 μm) were cut at a Leica CM 1860 UV cryostat and labelled with primary antibodies overnight at 4 °C. The following primary antibodies were used: monoclonal anti-NeuN (Millipore, USA) rabbit anti-PSD-95 (Cell Signaling Technology, Danvers, MA) and monoclonal anti-synaptophysin (BD Transduction Laboratories, Franklin Lakes, NJ). Primary antibodies were revealed with secondary antibodies coupled to Alexa Fluor® 488 and Alexa Fluor® 546 (Invitrogen), diluted 1:250 PBS (45 min, 37 °C). Sections were counterstained with Hoechst 33258; the dye, which binds specifically to A–T base regions in DNA and emits blue immunofluorescence at 350 nm, was administered at 1 ng/ml for 1 minute. Sections were observed at an Eclipse 80i Nikon Fluorescence Microscope (Nikon Instruments, Amsterdam, The Netherlands), equipped with a Video Confocal (ViCo) system.

For immunocytochemical characterization, retinal cell cultures were immunolabeled overnight at 4 °C with monoclonal anti-synaptophysin purchased from BD Transduction Laboratories (Franklin Lakes, NJ, USA), monoclonal anti-CRALBP from Santa Cruz Biotechnology (Dallas, TX, USA), rabbit polyclonal anti-GABA from Sigma (St. Louis, MO, USA), monoclonal anti-rhodopsin from Millipore (Billerica, MA, USA). Primary antibodies were revealed with secondary antibodies coupled to Alexa Fluor^® 488 or Alexa Fluor^® 546 (Molecular Probes, Eugene, OR, USA) (45 min, 37 °C). Nuclei were occasionally stained with Hoechst 33258. Immunostained retinal cultures were observed at the Eclipse 80i Nikon Fluorescence Microscope equipped with a ViCo system (Nikon, Tokyo, Japan).