Alexa fluor 488 and 555

siRNA-transfected HaCaT cells were rinsed in PBS at 37 °C, fixed in cold methanol for 3 min at −20 °C, blocked for 30 min at room temperature using PBS containing 2% BSA and 2% normal goat serum, and incubated with the following antibodies at the indicated dilutions: rat anti-DSC1 at 1:100 (MAB7367; R&D Systems, Inc., Minneapolis, MN, USA), rabbit anti-BubR1 at 1:100 (612503; BD Biosciences, San Jose, CA, USA). Cells were rinsed in PBS at 37 °C, fixed in 4% paraformaldehyde for 15 min at 37 °C, permeabilized for 5 min at room temperature using PBS containing 0.2% Triton X-100, and blocked for 30 min at room temperature using PBS containing 2% BSA and 2% normal goat serum. Secondary antibodies conjugated to Alexa Fluor 488 and 555 (Molecular Probes) were used at 1:2,000 dilution. After being washed in PBS containing 4,6-diamidino-2-phenylindole (DAPI) for 5 min, the coverslips were mounted in ProLong Gold (Thermo Fisher Scientific Inc). Fluorescence image acquisition was performed using a Nikon A1R confocal imaging system controlled by Nikon NIS Elements software (Nikon). The objective lens was an oil immersion Plan-Apo ×60 numerical aperture 1.40 lens (Nikon). Images were acquired as Z-stacks at 0.2-μm intervals, and maximum-intensity projections were generated using NIS Elements.

The brains of E20 (for CD31, MMP2 and MMP9), P0 (for hematoxylin and eosin and collagen IV), or P52 (for Luxol Fast Blue, myelin basic protein and SMI-312) rats that had been perfused with 10% neutral buffered formalin were post-fixed 2–3 days in paraformaldehyde. Brains were transferred to 30% sucrose for cryopreservation then frozen in OCT. Cryosections (10 μm) were stained with hematoxylin and eosin (H&E) or Luxol fast blue (LFB) following standard protocols [28 (link)].
Immunohistochemistry was performed as described [20 (link),28 (link)]. Sections were incubated overnight with primary antibodies, including: goat anti-CD31 (PECAM-1) (1:200, sc-1506; Santa Cruz Biotechnology, Santa Cruz, CA); rabbit anti-MMP2 (1:100; sc-10736; Santa Cruz); rabbit anti-MMP9 (1:200; #ab38898; Abcam, Cambridge, MA); rabbit anti-collagen IV (1:400; #ab6586; Abcam); rat anti-myelin basic protein (MBP) (1:200; #ab7349; Abcam); mouse anti-SMI-312 (1:200; #837901; BioLegend, San Diego, CA) at 4°C. After several rinses in PBS, sections were incubated with species-appropriate fluorescent secondary antibodies (Alexa Fluor 488 and 555, Molecular Probes, Invitrogen, Carlsbad, CA) for 1 hour at room temperature. Controls included the omission of primary antibodies.

WJ-MSC or HUVEC monolayers were grown on Lab-Tek® chamber slides with cover (Nunc, Naperville, IL, USA) up to 80% confluence, then rinsed in Hanks’ solution and fixed in 4% paraformaldehyde (15 min). Fixed cells were rinsed with Hanks’ solution, permeabilized with 0.1% Triton X-100 (20 min), and blocked (1 h) with 1% bovine serum albumin (BSA). Monolayers were incubated for 30 min at 37 °C with combinations of the following antibodies: Netrin-1 and −4 (R&D Systems), Neogenin-1 and eNOS (Santa Cruz Biotechnology, Dallas, TX, USA), UNC5b (Cell Signaling, Danvers, MA, USA), CD29 (Thermo Fisher Scientific), VEGF (Abcam, Cambridge, MA, USA) or CD31 (Sigma-Aldrich) followed by incubation with the corresponding secondary antibodies (Alexa Fluor 488 and 555; Molecular Probes, Eugene, OR, USA). Nuclei were stained with 4’, 6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich). Fluorescent secondary antibodies were visualized with Zeiss LSM 510 META (Carl Zeiss Microscopy GmbH., Jena, Germany).