Alexa flour 488 donkey anti rabbit

Mice underwent transcardiac perfusion on day 3 after SAH and brains were stored and fixed in 4% paraformaldehyde. Frozen brain samples were sectioned into 30 μm coronal sections using microtome. Brain slices were then incubated with a blocking buffer (0.4% Triton X-100 TBS-T with 5% donkey serum) for 2 h at room temperature and then incubated with primary antibodies (rabbit anti-NF-kB, #8242, Cell Signaling, Danvers, MA, USA {1:100}; Goat Anti-Iba-1, ab5076, Abcam, MA, USA {1:500}; Goat Anti-GFAP, A-31553, ThermoFisher, Waltham, MA, USA {1:200}; Rat anti-CD68, MCA1957, BIO-RAD, CA, USA {1:500}), followed by incubation at room temperature with secondary antibodies (Donkey anti-Rabbit Alexa Flour 488, A32790, ThermoFisher {1:1000}; Donkey anti-Goat Alexa Fluor 647, A-21447, ThermoFisher {1:1000}; Donkey anti-Rat Alexa Fluor 488, A-21208, ThermoFisher {1:1000}) in TBS-T for 45 min. After rinsing, the brain sections were stained with DAPI and mounted on the slide. The images were taken using Nikon Confocal microscopy. The fluorescent signaling was analyzed using Image J.