Spe confocal microscopy

To characterize the alignment of neurite outgrowths, retinoic acid-treated CE1 cells were cultured on fibers or control PLGA membrane and fixed as above. Anti-neurofilament (1:200; Santa Cruz Biotechnology) with Dylight 649 conjugated donkey anti-mouse secondary antibodies were used to define neurites outgrowth. The samples were observed and imaged by Leica SPE confocal microscopy. The alignment value was used to determine the alignment of neurite outgrowth, which was determined by the angle between the neurite outgrowth (neurofilament positive neurites) and the microfibers. The linear measurement tool of the ImageJ software was used to determine the alignment value of neurofilament-positive cells (n = 12 samples per group, 8–10 neurons were randomly selected per sample; Supplemental Fig. S1a).

The tertiary AC spheres were treated with 5-ethynyl-2-deoxyuridine (EdU, 200 ng/mL, Sigma) for 48 h as previously reported [28 (link),29 (link),34 (link)]. The spheres were fixed by 4% paraformaldehyde at room temperature for 10 min, followed by incubation in Click-iT reaction buffer, CuSO4, Alexa Fluor 555, and reaction buffer additive (Invitrogen) for 30 min. All nuclei were labeled with Hoechst 33342 (nuclei marker). The sphere samples were observed and imaged using Leica SPE confocal microscopy.

To characterize differentiation, retinoic acid-treated CE1 cells were cultured on either ACMFP fibers or flat PLGA membrane (control) for 6 days as described above. Cells were fixed in 4% paraformaldehyde, blocked with donkey serum in 0.5% Triton X-100 for 30 min, incubated with primary antibodies at 4 °C overnight followed by secondary antibody treatment at room temperature for 2 hr. Primary antibodies used in this study included: Anti-Sox2 (1:200; Abcam), Anti-Nestin (1:200; Developmental Studies Hybridoma Bank), Anti-TUJ1 (1:500; AVES), Anti-GFAP (1:200; Santa Cruz Biotechnology) and Anti-MOG (1:200; Millipore) antibodies. Secondary antibodies included Dylight 549 or Dylight 649 conjugated donkey anti-mouse, rabbit or chicken antibodies (Jackson Immunoresearch). 4,6-Diamidino-2-Phenylindole (DAPI; 1:500; Invitrogen) was used to label all nuclei. Samples were observed and imaged by Leica SPE confocal microscopy and/or Leica DMI 3000B epifluorescence microscopy. Cells cultured on coverslips serve as controls. The number of Sox2 and Nestin-positive cells, TUJ1 positive cells, GFAP positive cells and MOG positive cells were quantified via the Cell Counter plugin of the ImageJ software), and (Sox2 and Nestin double-labeled cells)/(DAPI positive cells) × 100% were calculated (n = 12 samples per group, 1–2 images were randomly selected per sample).

Immunofluorescence was performed on CD41⁺ cells cultured with or without inhibitors^{4 (link)}. In brief, cells were allowed to adhere on poly-l-lysine-coated slides for 1 h at 37 °C, fixed in 4% PFA, permeabilized with 0.2% of Triton, and blocked with 1% BSA before antibody labeling. Cells were examined under a Leica-SpE confocal microscopy with ×63/1.4NA oil objective. The following antibodies were used: mouse anti-ac-tubulin (1:300, Sigma T7451), mouse anti-Tubulin (1:100, Sigma T5293), rabbit anti-HDAC6 (1:100, Cell Signaling 7558), and rabbit anti-Cortactin (1:100, Millipore 05-180). FITC-conjugated phalloidin (1:500, Sigma P5282) was used to stain actin cytoskeleton, mouse anti-CD63 (1:100, Sigma SAB4700215, clone MEM 259), and rabbit anti-VWF (1:1000, Dako A0082). Appropriate secondary antibodies conjugated with Alexa 488, Alexa 546, or Alexa 633 (Molecular Probes) were used. DAPI (Molecular Probes) was applied for nucleus staining. Three-dimensional image analyses were performed with Zeiss Image Examiner software.

After fixation with 4% paraformaldehyde, cell samples were treated with PBS containing 5% donkey serum and 0.2% Triton-X100 for 20–30 min at room temperature. Samples were incubated in the primary antibody at 4 °C overnight, followed by corresponding secondary antibody incubation at room temperature for 2 hr. Secondary antibodies included AMCA, Alexa Fluor 488, Cy3, and Alexa Fluor 647 conjugated donkey anti-mouse, goat, rabbit, or chicken antibodies (Jackson Immunoresearch). 4,6-Diamidino-2-Phenylindole (DAPI; Invitrogen) was used to label all nuclei. Samples were observed and imaged by Leica 3000B epifluorescence microscopy and/or Leica SPE confocal microscopy.
For western blotting, purified TSP1 protein, ACM collected from wild type Swiss Webster mice, TSP1-ID-ACM, mock-ID-ACM, TSP1-KO-ACM, and DMEM/F12 were loaded to blotting gels. Antibodies used in western blotting included goat anti-TSP1 antibodies, donkey anti-goat HRP-conjugated antibodies, and HRP standard protein (all from Bio-Rad). SuperSignal West Femto Stable Peroxide Solution and SuperSignal West Femto Luminol/Enhancer Solution (ECL, all from Thermo scientific) were applied to blotting membrane for protein detection. Images were captured using a ChemiDoc-It 2 imaging system (UVP).

We incubated Hep G2 cells with 10 μg/ml DiI-LDL in medium for 2 h. After incubation, we washed the cells three times with DPBS+ 0.3% BSA and then fixed with 4% paraformaldehyde (Sigma Aldrich). We analyzed all samples using the Leica SPE confocal microscopy and analyzed the fluorescence signal using ImageJ software (https://imagej.nih.gov/ij/).