Dapi counterstain

Freshly dissected whole brain was snap frozen and cryosectioned in serial 30 µm sections. Sections were fixed with 10% buffered formalin, washed 3 times in 1XPBS and block in 2% cold water fish gelatin (Sigma, Inc) in 0.2% triton for one hour. Sections were then exposed to rat anti-dectin-1 and rabbit anti-CD45 (Cell signaling, Inc) antibody (1:100) in block overnight, washed with 1XPBS then treated with anti-rat alexFluor594 and anti-rabbit 488 for one hour. Sections were further washed in 1X PBS then mounted in media with DAPI counter-stain (SouthernBiotech). Images were acquired using a Zeiss 880 confocal microscope (Carl-Zeiss, Oberkochen, Germany).

Coronal sections were air-dried, washed with 1X PBS, blocked with 2% cold water fish skin gelatin (Sigma, Inc., St. Louis, MO, USA) in 0.2% triton-X100 for 1 h, incubated with a primary antibody diluted in blocking solution overnight at room temperature (RT), and then washed with 1X PBS six times 10 min each and incubated with appropriate secondary antibody solution (1:250 dilution) for 1 h at RT. Slides were then washed with 1X PBS and then mounted with media-containing DAPI counterstain (SouthernBiotech, Birmingham, AL, USA). Antibodies used are goat anti-EphA4 at 1:100 dilution (R&D systems, Minneapolis, MN, USA), rabbit anti-TMEM119 at 1:100 dilution (Abcam, Waltham, MA, USA), rabbit anti-CCR2 at 1:100 dilution (Abcam, Waltham, MA, USA), rat anti-CD68 at 1:100 dilution (Thermofisher, USA), rabbit anti-Iba1 at 1:200 dilution (FUJIFILM Wako Chemicals U.S.A. Corporation), donkey anti-goat 594 (Thermofisher, USA), donkey anti-rabbit 647 (Thermofisher, USA), donkey anti-rabbit 555 (Thermofisher, USA), and donkey anti-rat 647 (Thermofisher, USA). Image acquisition was performed using a Zeiss 880 confocal microscope (Carl-Zeiss, Oberkochen, Germany).

Perfused fixed brains were cryopreserved in 25% sucrose and then embedded in OCT and serial cryosectioned. Infarct volume (mm³) was assessed by the Cavalieri Estimator probe using the StereoInvestigator software (MicroBrightField, Williston, VT, USA), as previously described. Briefly, six serial coronal sections, cut at 30 μm, were stained using a 0.2% Cresyl violet solution (Electron Microscopy Science, Hatfield, PA) or anti-mouse IgG-488 (Invitrogen, Waltham, MA). The total volume of infarct was quantified by estimating the area of tissue loss in the ipsilateral cortical hemisphere using six, serial coronal sections. A 100 μm spaced grid was placed over the ipsilateral hemisphere in the Cavalieri probe and infarcted area scored. The coronal sections were used for immunohistochemical (IHC) staining of CD31. Sections were blocked with 2% cold water fish skin gelatin (Sigma, Inc., St. Louis, MO) in 0.2% Triton-X100, incubated with goat anti-CD31 (1 : 100, R&D systems, Minneapolis, MN, USA) overnight at room temperature (RT), washed with 1X PBS, and then incubated with donkey anti-goat 488 (1 : 250, Thermofisher, USA) for 1 hour at RT. Slides were then mounted with DAPI counterstain (SouthernBiotech, Birmingham, AL), and images were acquired using Olympus fluorescence microscope. Microvessel diameters were measured using ImageJ.

The freshly dissected whole brain was snap-frozen and cryosectioned in serial 30-μm sections. Sections were fixed with 10% buffered formalin, washed 3 times in 1× PBS, and blocked in 2% cold water fish gelatin (Sigma, Inc.) in 0.2% triton for 1 h. The sections were then exposed to mouse anti-EphA4 (ThermoFisher, Cat #:371600) antibody (1:100) in block overnight, washed with 1× PBS then treated with anti-mouse alexFluor594 for 1 h. The sections were further washed in 1× PBS then mounted in media with DAPI counterstain (SouthernBiotech). Images were acquired using a Zeiss 880 confocal microscope (Carl-Zeiss, Oberkochen, Germany).

TUNEL staining of serial coronal sections of perfused fixed brains was performed using the Click-iT Plus TUNEL Assay; 647 (Thermo Fisher Scientific) according to the manufacturer’s instructions. Serial coronal sections (300 μm) were permeabilized with 0.2% Triton X-100 in phosphate-buffered saline (PBS) and incubated with the TUNEL reaction mixture containing terminal deoxynucleotidyl transferase enzyme followed by Alexa Fluor 647-conjugated nucleotides. Following the TUNEL reaction, the sections were blocked in 2% cold water fish gelatin (Sigma, Inc.) in 0.2% triton for 1 h, then incubated with Rb anti-IBA1 (Wako) or Rb anti-NeuN (cell signaling) antibody (1:200 in blocking solution) overnight. Slides were then washed with 1×PBS, treated with AlexFluor donkey anti-rabbit-555 (1:250 in block) for 1 hour, further washed in 1×PBS, and then mounted in media with DAPI counterstain (SouthernBiotech). Z-stack images were acquired using a Zeiss 880 confocal microscope (Carl-Zeiss, Oberkochen, Germany).

Serial coronal sections of perfused fixed brains were blocked in 2% cold water fish gelatin (Sigma, Inc.) in 0.2% triton for 1 hour, incubated with AlexFluor donkey anti-mouse-594 (1:250 in block) for 1 hour, washed in 1×PBS, and then mounted in media with DAPI counterstain (SouthernBiotech). The volume of IgG deposition (mm³) was measured using the Cavalieri Estimator from StereoInvestigator software.