Immunostaining of Murine Brain Sections

Mice were anesthetized using isoflurane overdose and then perfused with 20 mL ice-cold 1× PBS, followed by 50 mL 4%PFA. Brains were postfixed overnight in PFA and then cryopreserved and sectioned as previously described (14 (link)). The 30 μm serial sections were blocked in 2% cold water fish gelatin (MilliporeSigma.) in 0.2% Triton for 1 hour and then incubated with Gt Anti-Tie2 (R&D Systems) Ab (1:200), Rb anti-Iba1 (Wako) Ab (1:200), Rb anti-Ccr2 (Abcam) Ab (1:200), and/or Gt anti-arginase-1 (1:500) in block overnight; washed with 1× PBS; and then treated with appropriate secondary Abs (Invitrogen): Alexa Fluor donkey anti-rabbit 594, Alexa Fluor donkey anti-rabbit 647, Alexa Fluor donkey anti-goat 594, and Alexa Fluor donkey anti-goat 647 (1:250 in block) for 1 hour. The sections were washed and mounted in media with DAPI counterstain (SouthernBiotech). Images were acquired using a Zeiss 880 confocal microscope (Carl-Zeiss).