Rabbit polyclonal anti neurofilament

Neuromuscular junctions (NMJs) were analyzed in the QL muscle. Muscles from P11 mice from each genotype were immersion fixed in 4% PFA for 20 mins and washed in PBS. Single fibers were teased using fine forceps and washed for 30 mins in PBS supplemented with 0.1M glycine. To stain the postsynaptic part of neuromuscular endplates fibers were incubated with alpha-bungarotoxin-555 antibody for 20 mins and washed in PBS before permeabilization with ice-cold methanol at −20°C for 2 mins. Fibers were then washed in PBS and incubated in a blocking solution containing 10% Donkey Serum in 0.3% PBS-T for an hour before exposure to antibodies against neurofilament - rabbit polyclonal anti-Neurofilament (1:500; Millipore) and synaptophysin - guinea pig polyclonal anti-synaptophysin (1:500; Synaptic Systems, 101 004) at 4°C overnight. Samples were washed in PBS prior to one-hour long incubation with the following secondary antibodies: Alexa Fluor 488 donkey anti-rabbit (Jackson ImmunoResearch, 711–545-152) and Donkey anti-guinea pig-Cy5 IgG (Jackson ImmunoResearch, 706–175-148). Fibers were washed and mounted in 30% glycerol in PBS.

Paraffin-embedded post-mortem brain tissue samples from healthy and HIV-infected individuals were provided by National NeuroAIDS Tissue Consortium (NNTC) and processed as described before (Zenon et al, in press). Mouse monoclonal anti-cathepsin B (1:100; Sigma-Aldrich), mouse monoclonal anti-SAPC (1:50; Abcam), rabbit polyclonal anti- ionized calcium-binding adapter molecule 1 (Iba-1) (1:100; Wako), mouse monoclonal anti-MMP-9 (1:65; R&D Systems), rabbit polyclonal anti-amyloid beta_1-42(Aβ) (1:50, Abcam), and rabbit polyclonal anti-neurofilament (1:100; Millipore, Billerica, MA) primary antibodies were incubated overnight at room temperature. Alexa Fluor® anti-mouse 488 and anti-rabbit 546 fluorescent secondary antibodies (1:200; Life Technologies) were incubated at room temperature for two hours. All sections were labeled with DAPI (1:500), diluted in Vectashield® (Vector Laboratories, Burlingame, CA). Images were acquired using a Nikon Eclipse E400 fluorescence microscope with a SPOT Insight QE camera and SPOT 5.1 software. A minimum of three images were acquired from each section.