Synaptophysin antibody

PSD-95 antibody (1:1,000, Cell Signaling, Danvers, MA) was used to detect PSD-95 (95 kDa). Synaptophysin antibody (1:1,000, Abcam, Cambridge, MA) was used to detect synaptophysin (38 kDa). Antibody anti-β-Actin (1:10,000, Sigma) was used to detect β-Actin (42 kDa). Western blot quantification was performed as described by Xie et al. (2008 (link)). Briefly, signal intensity was analyzed using ChemiDoc XRS+ with Image Lab 5.0 software (Bio-Rad, Hercules, CA). We quantified Western blots in two steps. First, we used β-Actin levels to normalize (e.g., determine the ratio of PSD-95 to β-Actin amount) the protein amount and to limit the disparities in the protein quantity loaded. Second, we presented the protein amount in the anesthesia/surgery treatment group as a percentage of those in the control group for comparison.

Cultured hippocampal neurons were fixed in 4% formaldehyde for 20 min, permeabilized for 6 min in 80% ice-cold methanol, and incubated with primary antibodies at 4 °C overnight. Primary antibodies used were: rabbit polyclonal tau antibody K9JA (1:1000; DAKO, Carpinteria, CA) and mouse monoclonal MAP2 antibody AP20 (1:1000; Sigma, St. Louis, MO) to assess tau missorting; mouse monoclonal phospho-tau antibody AT8 (1:500; Thermo Fisher Scientific, Waltham, MA) and rabbit polyclonal T22 (1:500; EMD Millipore) to measure tau phosphorylation and tau oligomer formation; rabbit polyclonal synaptophysin antibody (1:300; Abcam, Cambridge, MA); MC-1 antibody which detects misfolded murine tau^{116 (link)}; rabbit polyclonal tau antibody K9JA and mouse monoclonal α-tubulin antibody (1:1000; Sigma) to visualize neuritic beading. Other antibodies used for immunofluorescence included: mouse monoclonal drebrin antibody (1:300; Enzo, Farmingdale, NY), rabbit polyclonal p-cofilin^S3 antibody (1:300; Abcam), rabbit polyclonal p-Fyn^Y416 antibody (1:300; Cell Signaling, Beverly, MA); rabbit polyclonal p-NR2B^Y1472 antibody (1:600; Sigma) and mouse monoclonal p-tau^Y18 antibody (1:300; Medimabs, Montréal, QC). Secondary antibodies consisted of either donkey anti-rabbit or anti-mouse conjugated with either FITC or Cy3 (1:400; Jackson ImmunoResearch Laboratories, Inc. West Grove, PA).