Mouse anti neurofilament nf

A series of sections were selected for immunofluorescence to evaluate neuronal loss and gliosis in the CST zone and ventral horn. Briefly, sections were treated for 5 min with hot citrate buffer (85 °C, 0.01 m/L, pH 6.0) for post-antigen retrieval and then permeabilized with 0.3% Triton for 30 min followed by blocking with 5% normal goat serum for 1 h at room temperature. Subsequently, sections were incubated with the following primary antibodies at 4 °C overnight: mouse anti-neurofilament (NF, a marker of axons, 1:800, Sigma), mouse anti-neuronal nuclei (NeuN, a marker of neurons, 1:500, Millipore), rabbit anti-ionized calcium-binding adapter molecule 1 (Iba-1, a marker of microglia, 1:500, Wako), or rabbit anti-glial fibrillary acidic protein (GFAP, a marker of astrocytes, 1:500, Millipore). Negative control sections were incubated with 0.01 M PBS (pH = 7.4) instead of a primary antibody. After being rinsed with 0.01 M PBS for 10 min 3 times, sections were incubated for 1 h at room temperature with secondary antibodies: Alexa Fluor 488-conjugated goat anti-mouse IgG or Alexa Fluor 555-conjugated goat anti-rabbit IgG (1:1000, Cell Signaling Technology). Fluorescence signals were detected with a fluorescence microscope (BX51, Olympus).

Immunohistochemical staining was performed using the Dako autostainer platform (Dako, Denmark) as previously described [24 (link)]. Sections were stained using the following primary antibodies: mouse anti-CD68 (clone PG-M1, 1:100, Dako), mouse anti-CD45 (clone 2B11, 1:200, Dako), rabbit anti-Iba1 (ionized calcium binding adaptor molecule 1, 1:1000, Wako), rabbit anti-GFAP (1:2000, Dako), mouse anti-neurofilament (NF) (phosphorylated and non-phosphorylated NF-heavy chain; clone N52, 1:1000, Sigma-Aldrich), mouse anti-IL-1α (clone 4414, 1:1200, R&D Systems), mouse anti-IL-1β (clone 2E8, 1:50, BioRad), rabbit anti-TNF (1:100, ThermoFisher Scientific), rabbit anti-TNFR1 (clone H-271, 1:50, Santa Cruz), rabbit anti-TNFR2 (1:50, Sigma-Aldrich), and rat anti-IL-1Ra (clone 40,007, 1:1500, R&D Systems). The antigen-antibody complex was visualized using EnVision+System horse-radish peroxidase-labelled Polymer (Dako), PowerVision+Poly-HRP IHC (AH Diagnostics), or CSAII (Dako) detection systems.

The following antibodies were used as primary antibodies for immunocytostaining and for immunohistochemistry: rabbit anti‐s100b (dilution = 1:1,000) (Dako, Ely, UK), rabbit anti‐p75 (1:500) (Cell Signaling Technology, Danvers, MA, USA), rabbit anti‐GAP43 (1:200) (Cell Signaling Technology), rabbit anti‐glial fibrillary acidic protein (GFAP) (1:200) (Abcam, Cambridge, UK), rabbit anti‐NG2 (1:1,000) (Millipore, Cambridge, UK), mouse anti‐myelin basic protein (MBP) (1:100) (Chemicon, Chandlers Ford, UK), goat anti‐protein zero (P0) (1:500) (Abcam), rabbit anti‐Tuj‐1 (1:100) (Chemicon), mouse anti‐neurofilament (NF) (1:1,000) (Sigma), rabbit anti‐PGP9.5 (1:500) (Abcam), mouse anti‐fibronectin (Millipore), mouse anti‐Nanog (1:200) (ReproCELL, Yokohama, Japan), mouse anti‐MAP‐2 (1:200) (Abcam), and rabbit anti‐NeuN (1:500) (Abcam) antibodies. As secondary antibodies, Alexa Fluor 546‐conjugated anti‐rabbit and anti‐mouse antibodies and Alexa Fluor 670‐conjugated anti‐rabbit and anti‐mouse antibodies (1:1,000) (Life technologies, Carlsbad, CA, USA) were used.