Anti rabbit iba 1

Mice were perfused with saline solution and formalin, and then tissues were dehydrated with 30% sucrose in PB solution at 4°C for 3‐4 days. Immunostaining was performed on free‐floating 20‐µm sections. Brain sections were blocked in 10% donkey serum for 1 hour and 1% Triton X‐100 in PBS for 20 minutes at room temperature and probed with the following primary antibodies overnight at 4°C: antimouse iNOS (BD, 610329), antirabbit iba‐1 (Wako, 019‐19741), antigoat Arg‐1 (Santa Cruz, F0915), and antimouse‐NeuN (MAB377,4739). After they were washed, the sections were treated with FITC‐labeled Alexa Fluor‐488‐ and/or Alexa Fluor‐594‐conjugated secondary antibody at a 1:1000 dilution for 1 hour. The sections were covered with DAPI Fluoromount‐G® (Southern Biotech). The stained cells were visualized and photographed by a confocal microscope.

The processed brains were embedded in paraffin, and 5-µm coronal sections were prepared for every 1 mm. For immunohistochemical detection, anti-mouse GFAP (glial fibrillary acidic protein, a marker of astrocytes) antibody (1:100; Merck Millipore, Burlington, VT, USA), and anti-rabbit Iba1 (ionized calcium binding adaptor molecule, a marker of microglia 1) antibody (1:200; WAKO, Osaka, Japan,) were used. The secondary antibody was a goat-anti-mouse IgG polyclonal antibody (Nichirei Bioscience Inc., Tokyo, Japan). Slices were stained with 3,3′-diaminobenzidine (DAB) with hydrogen peroxide. The GFAP-positive astrocytes and Iba1-positive microglia were segmented by applying an appropriate threshold in gray value in order to distinguish from non-specific background staining. The percent areas (segmented area/total 400 μm × 400 μm area) were calculated using ImageJ software. The regions of interest (ROIs), i.e., square frame, were laid not to include tissue absent area or necrotic area by an examiner who was blinded to the experimental group of the mice. Four regions were determined from a section containing both the hippocampus and striatum; the peri-infarct cortex, hippocampus, subcortical white matter, and non-infarct cortex in an area of 400 μm × 400 μm. We calculated the percent area for two ROIs in one slice per region per animal (n = 5 per group).

Sprague Dawley cortices were digested and grown in DMEM containing 10% FBS with 4.5 g/l glucose, L-glutamine, pyruvate, and 1% penicillin/streptomycin (DMEM-10%) using standard methods [31 (link)]. After 7–10 days, microglia and OPCs were purified by differential attachment [32 (link)]. Microglia were plated in DMEM-10% on poly-L-lysine (PLL, Sigma, 13 µg/ml) coated glass coverslips (VWR, 10 µg/ml) at 5 × 10⁴ cells/coverslip. Microglia were stained using anti-mouse CD4 (1:100, ThermoFisher Scientific) and anti-rabbit Iba-1 (1:100, Wako) using standard immunohistochemistry methods described above. OPCs were plated onto 13 mm PLL coated glass coverslips at 4 × 10⁴ cells/coverslip in DMEM-BS containing FGF2 (50 ng/mL) and PDGF (50 ng/mL) for 5 days, then treated in duplicate with IL-16 (100 ng/ml) or DMEM-BS for 4 days. On day 5, cells were immunolabelled with anti-rabbit NG2 (1:100, Abcam), anti O4 (1:100, IgM, hybridoma) or anti-rat PLP (1:100, AA3, hybridoma) and 10 images taken per coverslip with 2 coverslips per treatment, with an average of 200–300 cells/coverslips analysed. OPCs were stained using CD4 described above and co-stained using anti-O4 (1:100, IgM, hybridoma).

After 1 week of OHSC preparation (wild-type––WT or microglia depleted––Lip-CL), OHSC were washed with DPBS followed by 4% paraformaldehyde (PFA) incubation for 1 h. After fixation, the slices were washed with DPBS and incubated with 5% normal goat serum (NGS - Vector) in DPBS containing 0.3% Triton X-100 (DPBS⁺) for at least 2 h. Subsequently, the slices were incubated overnight with mouse anti-GFAP (1:1000, Cell signaling), anti-rabbit-Iba-1 (1:1000, Wako), and DAPI (1:1000, Sigma) in 1% NGS/DPBS⁺ at 4 °C. Then, slices were incubated with the secondary antibodies for 2 h at room temperature. Rabbit highly cross-adsorbed AlexaFluor 594, and mouse AlexaFluor 488 secondary antibody (Invitrogen, Carlsbad, CA, USA) was used to detect Iba-1 or GFAP, respectively. Slices were imaged in a Zeiss microscope (Zeiss, Oberkochen, Germany).