Abc kit

Gas6, Mer, Axl, and Tyro-3 proteins in brain tissues were measured by immunohistochemistry of 5-μm tissue sections. Anti-Myelin Basic Protein antibody (MBP antibody sc-271524, 1:500, Santa Cruz Biotechnology, Dallas, TX, United States) labels myelin which is the most abundant protein in the myelin membrane. For this, sections were deparaffinized, rehydrated, and treated with 3% (v/v) H₂O₂ in methanol for 30 min, followed by blocking with 5% (w/v) fat-free milk for 1 h. Subsequently, they were incubated with a primary antibody (anti-Mer, ab79223; anti-Axl, ab219651; or anti-Tyro 3, ab109231; anti-FOXP3 antibody, ab20034, 1:100, Abcam, UK) at 4°C overnight. After washing, bound antibodies were detected with biotin-labeled secondary antibodies using the ABC kit, visualized with diaminobenzidine, and examined by light microscopy (Thermo Fisher Scientific, USA). Immunostained slides were independently scored by at least three investigators and the mean of the results was calculated.

The immunocytochemistry was performed using a previously described method [23 (link)]. Briefly, the cultured neurons were fixed with 4% paraformaldehyde and exposed to PBS (NaCl 8.5 g, Na₂HPO₄·12H₂O 7.05 g, and NaH₂PO₄·2H₂O 0.52 g dissolved in 1000 ml of distilled water, pH 7.4) containing 0.3% Triton X-100, followed by incubation in 5% BSA to block non-specific binding. The neurons were then incubated with a rabbit anti-MAP-2 antibody (1:1000) diluted in PBS at 4°C for overnight. After washing with PBS, the neurons were incubated with secondary antibody for 1 h at room temperature. Subsequently, the results were visualized using the ABC kit (Thermo Scientific, Grand Island, NY, USA) and analyzed using an inverted Nikon TE300 microscope at ×200 magnification. Ten areas were randomly captured from each well. The length of MAP-2 positive neurites of each neuron was measured by the NIH Image J software with the Simple Neurite Tracer plugin according to the manufacture’s instruction (http://imagej.net/Simple_Neurite_Tracer:_Step-By-Step_Instructions), whereas the neurons connecting with other neurons or with neurite endings outside the optic field were excluded. The total neurite length of all neurons counted in a group was divided by the total number of neurons, and then, the average of each group was normalized to that of the vehicle control.

Rats in each group were perfused at 6, 12, 24 h, 7 d after successful modeling. Rats were anesthetized with chloral hydrate at 350 mg/kg. The chest and abdomen were cut open to expose the heart and liver. The perfusion needle punctured through the left ventricular apex. Then the right atrial appendage was opened, followed by the rapid perfusion of 200 mL heparinized saline and then 200 mL paraformaldehyde (4%) until the rat limbs became straight and the liver turned white. The whole brain was removed, fixed in 4% paraformaldehyde for 12 h, and transferred to 20 and 30% sucrose overnight. The brain tissue was then cut into 10-μm thickness. Immunohistochemical staining was performed using the ABC kit (Thermo Scientific, USA). All procedures were conducted according to the manual instructions. The antibodies used are listed as follows: neuronal nitric oxide synthase (nNOS) (1:1,000, Chemicon), protein kinase C-delta (PKCδ) (1:300, Santa Cruz), vascular cell adhesion molecule-1 (VCAM-1) (1:50, Sigma), intercellular adhesion molecule-1 (ICAM-1) (1:50, Sigma), active caspase-3 (1:10, Chemicon). Notably, all stainings were performed at the same time point.