Anti ionized calcium binding adaptor molecule 1 iba 1

Dulbecco’s modified Eagle’s medium: Nutrient Mixture F-12 (DMEM/F12), Neurobasal Medium, B-27 supplement, phosphate-buffered saline (PBS) buffer, fetal bovine serum (FBS) and anti-glial fibrillary acidic protein (GFAP) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Recombinant mouse epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) were purchased from R&D Systems (Minneapolis, MN, USA). Poly-L-ornithine (PLO), laminin and bovine serum albumin (BSA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-Nestin and oligodendrocyte marker O4 (O4) antibodies were purchased from Millipore (Burlington, MA, USA). Anti-Sox2 and Ki67 antibodies were purchased from Abcam (Cambridge, UK). Anti-ionized calcium-binding adaptor molecule 1 (Iba1) was purchased from FUJIFILM Wako Chemicals (Richmond, VA, USA). Growth-factor-reduced Matrigel was purchased from BD Biosciences (Bedford, MA, USA). Alexa 568 goat anti-mouse, Alexa 568 goat anti-rat, Alexa 488 goat anti-rabbit, and Alexa 488 and 568 goat anti-rabbit secondary antibodies were purchased from Life Technologies (Carlsbad, CA, USA). WKYMVm (Trp-Lys-Tyr-Met-Val-D-Met-NH₂) was purchased from Anygen Inc. (Gwangju, Korea).

Animals were sacrificed at the indicated times after surgery. Brains were isolated and fixed with 4% paraformaldehyde (PFA; Sigma Aldrich, St. Louis, MO) by transcardiac perfusion and then stored in the same solution overnight at 4 °C. Brain sections (40 μm) were prepared using a vibratome and immunological staining was performed using a previously described floating method [13 (link)]. The sections were preincubated in blocking solution containing 5% FBS, 5% horse serum, and 2% BSA in PBS. Primary antibodies were diluted 1:500 for anti-ionized calcium binding adaptor molecule-1 (Iba-1) (Wako Pure Chemicals, Osaka, Japan). After washing with PBS containing 0.1% Triton X-100, sections were incubated with anti-rabbit IgG (Vectorlab, Peterborough, UK) in PBS for 1 h at room temperature and visualized using the HRP/3,3-diaminobenzidine (DAB) system and observed under a fluorescence microscope (Axioplan 2; Zeiss, Oberkochen, Germany).

We used 0.01 M PBS in every procedure. After washing with 0.01 M PBS, the retinal sections were blocked in 10% normal donkey serum for 1 h with 0.1% Triton X-100. Subsequently, they were incubated with primary antibodies for 18 h at 4 °C. The sections were washed in PBS and incubated with a secondary antibody for 2 h at room temperature. Cell nuclei were counterstained with DAPI for 5 min. Anti-GRP78 (1:1000; Abcam, Cambridge, UK), anti-ionized calcium binding adaptor molecule 1 (IBA1) (1:500; Wako Chemical, Osaka, Japan), anti-glutamine synthase (GS) (1:1000; Chemicon, Temecula, CA, USA), anti-glial fibrillary acidic protein (GFAP; 1:1000; Chemicon) and Cy3 (1:1500; Jackson Immunoresearch, West Grove, PA, USA) or Alexa 488-conjugated antibodies (1:1000; Molecular Probes, Eugene, OR, USA) were used as secondary antibodies. Images were obtained using a Zeiss LSM 800 confocal microscope (Carl Zeiss Co. Ltd., Oberkochen, Germany).