Monoclonal anti neun

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.

The following primary antibodies were used: polyclonal anti-SVIP, monoclonal anti-NeuN (Millipore), monoclonal anti-CNS (Millipore), monoclonal anti-GFAP (Beyotime Bio, Shanghai, China), and monoclonal anti-CD11b/integrin am (OX-42). Polyclonal anti-β-actin, polyclonal anti-p53, polyclonal anti-Ki67, polyclonal anti-PSA (A67-B/E3), and anti-AR (N-20) antibodies were obtained from Santa Cruz (TX, USA). Control siRNA and siRNA targeting human SVIP have been previously described [36 ], siRNAs targeting SVIP (5′→3′): siRNA #1, GACAAAAAGAGGCTGCATC; siRNA #2, CCCTTA AGTGCAATGCTAA; siRNA #3, GACAGTTTCATAA AGCATA.

Immunodetection of BrdU and neuronal markers in tissue sections was carried out as previously described (Abbott et al., 2013 (link)). Primary antibodies used were: rat anti-BrdU (Abcam), rabbit anti-Doublecortin (Cell Signaling Technology Inc., Beverly, MA, USA), monoclonal anti-NeuN (Millipore, Billerica, MA, USA) and rabbit anti-Ki67 (Abcam). As secondary antibodies, Alexa (Molecular Probes) and DyLight (Abcam) conjugated antibodies were used. BrdU and Ki67 positive cells were counted using a fluorescence microscope (Olympus BX51, Tokyo, Japan) as described (Abbott et al., 2013 (link)). Double-labeled sections were analyzed by confocal laser microscopy (Olympus FV 1000). Image analysis and z-projections were made with ImageJ software (NIH, USA).