Donkey alexa fluor 488

Cells were cultured on 8 chamber slides at a density of 1 × 10⁴ cells per well. After 21 days of culture in osteoblast differentiation cocktail, cells were fixed with a 95% ethanol and 5% acetic acid for 10 minutes at −20°C. After washing with PBS, cells were permeabilized with 0.1% (v/v) Triton X-100 (Sigma) for 45 minutes and washed again with PBS. Nonspecific binding was blocked by incubating in 1:20 horse serum (Sigma) for 1 hour. Cells were then incubated with mouse anti-human osteocalcin (R&D Systems, Minneapolis MN) at 4°C overnight. After washing with PBS, cells were incubated with secondary antibody (donkey Alexa-Fluor-488) (Invitrogen, Carlsbad CA) for 1 hour at room temperature. Nuclear counterstaining was performed with mounting medium containing DAPI (Vector, Burlingame CA).

Rats were euthanized on day 1 or day 3 of reperfusion by transcardiac 4% paraformaldehyde (PFA) perfusion fixation. Brains were post-fixed in 4% PFA, cryoprotected and sectioned (coronal: 40μm thickness). Brain sections were then immunostained with primary antibodies against REST (1:300; Proteintech), NeuN (1:300; Millipore), cleaved caspase-3 (1:400; Cell Signaling), and phosphorylated dynamin-related protein 1 (Drp1; 1:300; Cell Signaling) followed by donkey Alexa Fluor 488 or Alexa Fluor 594 secondary antibodies (1:300; Invitrogen).