Anti dcx

All samples were stained as previously described by our laboratory(Hill et al. 2018a (link); Hill et al. 2018b (link)). Antibodies used were anti-Ki67 (1:500, Abcam, Cambridge, UK; Cat# ab15580 Lot# GR264777 RRID:AB_443209), anti-DCX (1:1000, Millipore, Burlington, MA, USA; Cat# AB2253 Lot# 2828588 RRID:AB_1586992), anti-BrdU (1:200, Abcam, Cat# ab6326 Lot# GR267766–1 RRID:AB_305426), anti-IBA-1 (1:500, Wako Chemicals, Richmond, VA, USA; Cat# 019–19741 Lot#WDK2121 RRID:AB_839504), and anti-CD68 (1:1000, Bio-Rad Laboratories, Hercules, CA, USA; Cat# MLA1957 Lot#1708 RRID:AB_322219). Secondary antibodies used were Alexa-488® goat anti-guinea pig (Thermo Fisher Scientific, Waltham, MA, USA; Cat# A-11073 Lot#1841755 RRID:AB_2534117), Alexa-594® donkey anti-rat (Thermo Fisher Scientific; Cat# A-21209 Lot#45081A RRID:AB_2535795), Alexa-488® donkey anti-rabbit (Thermo Fisher Scientific; Cat# A-21206 Lot#1608521 RRID:AB_2535792). All secondary antibodies were used at a dilution of 1:400. Imaging for in vivo quantification was performed using a CoolSNAP EZ CCD camera (Photometrics, Tucson, AZ, USA) coupled to a Nikon i80 Eclipse (Nikon Instruments Inc., Melville, NY, USA). Confocal images were taken on a Nikon A1R confocal microscope (Nikon).

At different time points after stroke, mice were perfused transcardially
with a solution of 4% paraformaldehyde (PFA, pH 7.2) in 0.1 M
phosphate buffer (PB, pH 7.2). Brains were removed from the skull, post-fixed in
4% PFA overnight at 4 °C, and sequentially transferred to 20% and 30%
sucrose in 0.1 M phosphate buffer, pH 7.2 solutions overnight. Brains were
frozen on dry ice and sectioned on a cryostat to obtain coronal sections of 30
μm in thickness.
The sections were then incubated with blocking buffer for one hour. The
primary antibodies were prepared in the blocking buffer and the sections were
incubated in the solution overnight: anti-GFP (Green fluorescent protein)
(1:1000; Invitrogen) and anti-DCX (1:500, Millipore). After incubation with
primary antibody solution, the sections were washed and incubated for four hours
at room temperature in diluted secondary antibody prepared with blocking
solution (secondary antibody conjugated with Alexa 488 or Alexa
555, 1:1000; Life Technologies, Carlsbad, CA, USA). The slides were then washed
with PB solution and cover-slipped. Images were acquired using a Leica
microscope. Omission of primary or secondary antibodies resulted in no staining
and served as negative controls. Group and treatment information are all blinded
to image analyzer.