4 6 diamidino 2 phenylindole dapi dye

Heart sections were incubated with rabbit polyclonal antibody vWF (1:50 dilution, Millipore) over night at 4 °C after deparaffination, 3% H₂O₂ treatment and antigen retrieval. HRP-conjugated goat anti-rabbit IgG (R&D, Minneapolis, MN 55413) was used as a secondary antibody and diaminobenzidine (R&D) for color development. Nuclei were stained by hematoxylin. For immunofluorescent staining, sections were incubated with primary antibody over night at 4 °C after deparaffination and antigen retrieval. The dilutions of primary antibodies were as follows: rabbit polyclonal antibody FSTL1 (1:100 dilution, GeneTex), rabbit polyclonal antibody vWF (1:50 dilution, Millipore), mouse monoclonal antibody CD31 (1:25 dilution, GeneTex) and mouse monoclonal antibody PCNA (1:50 dilution, Cell Signaling). TRITC/FITC-conjugated goat anti-rabbit/mouse IgG (1:100 dilution, Jackson ImmunoResearch) were used as secondary antibodies. Nuclei were stained by 4′6-diamidino-2-phenylindole (DAPI) dye (1:800 dilution, Sigma, St. Louis, MO, USA). Results were observed with a fluorescence microscope (Nikon Eclipse 55i, Nikon, Tokyo, Japan).

For cellular adhesion identification, 2 × 10⁴ cell line G292 cells were seeded onto scaffolds and left for 18 h to adhere to the scaffold surfaces. Cell seeded scaffolds were washed twice with PBS, then fixed with 1% neutral-buffered formalin (NBF), permeabilised with 1 (v/v)% Triton X-100 (Sigma Aldrich, Germany) for 5 min and then washed twice using PBS. The cell-seeded scaffolds were incubated with Alexa Flour-488 phalloidin (Invitrogen, Waltham, MA, USA) for 2 h to stain the actin filaments and co-incubated with 4′,6-diamidino-2-phenylindole DAPI dye (Sigma-Aldrich) for 15 min to stain the cell nuclei. Stained cells were rinsed with PBS twice and then imaged using Leica TCS SP8 confocal microscope (Germany).

Each cell line was seeded in 96-well plates at 10⁴ cells. Once adhered, cells were treated with AKBA, ABA, BA, and DMSO as a negative control, with the various dosages. After 24 h of treatment, cell viability was evaluated using a 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay kit (Bio-idea, Tehran, Iran). The optical value was read by using of a BIO-RAD (Hercules, CA, USA) reader at a wavelength of 570 nm.
After 24 h of cells treatment with 65 μM AKBA, ABA, and BA, the viability was further assessed utilizing the live and dead assay kit (ThermoFisher Scientific, USA) according to the manufacturer's protocol. Using this assay, live cells were distinguished from the dead cells by fluorescence microscopy (Optika XDS 3FL4, Italy) with excitation and emission of green (ex/em 494/530 nm for Calcein AM) for live cells and red (ex/em 528/645 nm for EthD-1) fluorescence for dead cells^{50 (link)}. For nuclear staining, the 24 h-treated cells were stained with 4′,6-diamidino-2-phenylindole (DAPI) dye (Sigma-Aldrich, USA), following of rinsing the cells with PBS. Finally the cells were evaluated using a fluorescent microscope (Optika XDS 3FL4).

Antigen retrieval was performed by immersing sections in 10 mM of citrate buffer (pH = 6.0) at 90°C for 10 min using a pressure cooker. Brain sections were blocked in 1% bovine serum albumin in phosphate-buffered saline containing 1% Triton X-100 for 30 min, then incubated with neuronal marker chicken anti-neuronal nuclei (NeuN; 1:5000; ABN91; Merck Millipore) for ∼56 h and Alexa Fluor^TM 555 goat anti-chicken (1:1000; A11039; ThermoFisherScientific) overnight at room temperature. Sections were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) dye (1:1000; Sigma-Aldrich) for 5 min to stain the nuclei and mounted using DABCO (Sigma-Aldrich). Cells labeled with NeuN and DAPI in the ipsilateral cortex near the lesion region were measured using the Colocalization pipeline of CellProfiler Image Analysis software (Broad Institute, Cambridge, MA).