Primary rabbit anti iba 1 antibody

Microglia were stained using an antibody against the microglial marker, ionized calcium–binding adaptor molecule 1 (Iba-1) as described previously [17 ,32 (link)]. Briefly, the sections were firstly blocked with 10% bovine serum albumin for 1 h at room temperature. After that, the sections were incubated with a primary rabbit anti–Iba-1 antibody (1:800: Wako, 019–19741) at 4°C overnight. After incubation with an Alexa 488–conjugated donkey anti–rabbit IgG antibody (1:500; Molecular Probes, Rockford, USA) for 1 h, the stained sections were examined under a microscope (Carl Zeiss Axio Observer Z1, Jena, Germany) determined the number of immunoreactive cells in the medial superficial dorsal horn (laminae I–III). Nonspecific staining was determined by omitting the primary antibody. The data were calculated as average numbers of positive cells per area of a spinal section ± standard error of the mean (SEM).

Lumbar region of the spinal cords was used for immunofluorescence studies. For immunofluorescence, sections were incubated overnight at 4 °C with primary rabbit antibody against cleaved (activated) caspase 3 (Asp175; 1:300; Cell signalling, Danvers, MA, USA) or primary mouse anti-ED1 antibody (1:200, Abcam, Cambridge, MA, USA) or primary rabbit anti-Iba1 antibody (1:200, Wako, Richmond, VA, USA), then washed in PBS and incubated for 1h with appropriate Alexa Fluor 488- or Alexa Fluor 566-conjugated secondary antibodies (Molecular Probes, Inc. Eugene, USA). Negative controls were carried out by omitting the primary antibody. Sections were mounted with Vectashield with DAPI (Vectorlabs, Burlingame, CA, USA).
Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was performed using an in situ cell death detection kit (Fluorescein or TMR; Roche Diagnostics GmbH, Mannheim, Germany).