Ab9774

The following primary antibodies were used: Mouse monoclonal anti-RECA1 (Abcam, ab9774, 1:100) that recognizes a cell surface antigen expressed on the surface of rat endothelial cells. RECA1 is a pan-endothelial marker and labels both fenestrated and non-fenestrated vasculature. Fenestrated blood vessels were labeled with rabbit polyclonal antibody against plasmalemmal vesicle-associated protein 1 (PV1) provided by Dr. Philippe Ciofi (INSERM, Bordeaux, France, 1:250) (Ciofi et al., 2009 (link)). Tanycytes and ependymocytes were labeled with chicken polyclonal antibody against vimentin (Millipore, AB5733, 1:5,000). Vimentin is an intermediate filament that in adult tissue is primarily expressed by tanycytes and ependyma cells (Franke et al., 1978 (link)). Astrocytes were labeled with rabbit polyclonal GFAP (glial fibrillary acidic protein) antibody (Abcam, ab7260, 1:1,000), neurons with NeuN (Hexaribonucleotide Binding Protein-3a) guinea pig polyclonal antibody (Millipore, ABN90, 1:500), NG2 glia and pericytes with rabbit polyclonal antibody against the chondroitin sulfate proteoglycan NG2 (Millipore, AB5320, 1:500) and goat polyclonal anti-CD13 (R&D systems, AF2335, 1:100), and c-Fos with rabbit polyclonal antibody (SYSY, 226 003, 1:500). Secondary antibodies were Alexa Fluor-Conjugated (488, 568, and 647 nm; Invitrogen, 1:500).

According to a previous method [46 (link)], collagenase perfusion and percoll layers of different density gradients (25% and 50%) (GE Healthcare, MA, USA) were used to isolate LSECs. After harvested, they were seeded on 12 mm coverslips (Electron Microscopy Sciences, PA, USA) coated with type I collagen (Thermo Fisher Scientific, MA, USA) at a density of 2.5×10⁵ cells/cm². The purity of the cultured LSECs was > 90%, as determined by immunofluorescence assays with anti-von Willebrand factor (vWF) (11778-1-AP, Proteintech) and anti-endothelial cell antigen-1 (RECA-1) antibodies (ab9774, Abcam, UK) (Supplementary Figure 2).

Animals were deeply anesthetized with pentobarbital (50 mg/kg, i.p.) and transcardially perfused with cold 0.1 M phosphate buffer saline followed by cold 4% paraformaldehyde in 0.1 M phosphate-buffered saline. Brains were removed and immersed in 4% paraformaldehyde for 24 h and 30% sucrose for another 24 h. Coronal brain slices (20 μm thickness) were cut. Slices were collected at +1.0, 0.0, and −1.0 mm (center of the hemorrhagic lesion) anterior and posterior to bregma using a cryostat (Leica CM 1900) and processed for the staining and counting of marker-specific cells. The following primary antibodies were used, with the dilution rates and retrieval methods indicated in parentheses. Antibodies against CD91(1:500, Abcam, ab92544), CD36 (1:500, Novus, NB400-144), CD163 (1:250, Abcam, ab182422), CD11b (1:200, AbD Serotec; 1:250, Abcam, ab1211), CD68 (1:500; Bio-Rad, MCA341R) and Arginase-1 (1:500, Santa Cruz, sc-271430) were used as microglia/macrophage markers. Anti-RECA antibody (1:200, Abcam, ab9774) was used as an endothelial marker. Anti-GFAP antibody (1:500, NOVUS, NBP-05197) was used as a glial marker, and anti-NeuN antibody (1:1000; Chemicon, MAB377) was used as a neuron marker.

To evaluate the histology of healing tissue, retrieved bone specimens were fixed and decalcified before embedded in paraffin and sectioned longitudinally, which were stained with hematoxylin and eosin (H&E) and alcian blue. The histological staining was visualized and scanned using Panoptiq digital slide imaging system (ViewsIQ, Richmond, Canada).
For immunohistochemical staining, the paraffin-embedded specimen was sectioned and processed as previously described9 (link). Briefly, tissue slides were deparaffinized and rehydrated before antigen retrieval and blocking with goat serum. Then, the slides were incubated with primary antibodies (Ab), either mouse-anti-human osteocalcin Ab (ab13421, Abcam), mouse-anti-rat osteocalcin Ab (M186, Takara), rabbit-anti-rat collagen-II Ab (AB2036, Millipore) or mouse-anti-rat endothelial cells (ab9774, Abcam), before being incubated with secondary antibody conjugated with Alexa-Fluro-488 (A-11008, or A-11029, ThermoFisher Scientific). Finally, the tissue slides were covered by anti-fade mounting medium with DAPI (H-1200, Vector Laboratories) and visualized under Zeiss Axioplan fluorescence microscope (Carl Zeiss, NY).

Slides were washed with phosphate buffered saline (PBS) and then blocked with 1% v/v normal donkey serum (NDS; Sigma-Aldrich, Gillingham, Dorset, UK), 1% v/v bovine serum albumin (BSA; Sigma-Aldrich), and 0.1% Triton-X in PBS. Primary antibodies for endothelial cells [1:200 mouse anti-rat endothelial cell antigen-1 (RECA-1; ab9774, Abcam, Cambridge, UK)] and basement membrane [1:200 rabbit anti-laminin (L9393; Sigma-Aldrich)] were prepared in 0.1% Triton-X PBS (PBST), and sections incubated at 4°C overnight. Secondary antibodies conjugated to Alexa-fluor 488 [1:500 goat anti-mouse (A11001; Invitrogen, Waltham, Massachusetts, USA)] and Alexa-fluor 546 [1:500 goat anti-rabbit (A11010; Invitrogen)] were also prepared in PBST. After three washes with PBS, sections were incubated with secondary antibodies for 2 hours at room temperature. Sections were washed in Tris non-saline (TNS) and mounted with Vectashield^® containing DAPI (Vector Laboratories, Burlingame, California, USA). Four areas (ventral grey matter, ventral white matter, dorsal grey matter, dorsal white matter) of three replicate sections were imaged for all sections at all time points and spinal segments on a Nikon E600 microscope and analysed in FIJI (NIH ImageJ; Schindelin et al., 2012 (link)).

Slides were thawed and washed twice with PBS; blocked and permeabilized with PBS 1% bovine serum albumin, 5% goat serum (Biological Industries, Beit Ha’emek, Israel) and 0.05% Triton-X (Sigma-Aldrich) for one hour; and incubated with the following primary antibodies overnight at 4 °C: αSMA (1/400, 19245, Cell Signaling, Danvers, MA, USA), GFAP (1/1000, ab7260, Abcam), MPO (1/100, ab90810, Abcam), RECA (1/100, ab9774, Abcam). Slides were washed three times with PBS and incubated with a fluorescent-labeled secondary antibody (1/700, Alexa-Flour) for 1 h at RT. For IgG staining, brains were incubated overnight with 1/200 Alexa-Flour Cy3 anti-rat IgG antibody. DNA was stained for 10 min with DAPI (1:1000, Sigma-Aldrich). Slides were mounted with Flouromount-G. αSMA and RECA images were used to calculate vessel area. In order to minimize the background signal, slides were incubated with secondary antibodies and without primary antibodies. This ensures that staining is produced from the detection of the antigen by the primary antibody and not by the detection system or the specimen.