Hb050

HeLa and Caco-2 cells were seeded in 12-well flat-bottomed plates with sterile-covered round objects covered with collagenase at a density of 1 × 10⁴ cells per well with one mL of CM and incubated for 24 h at 37°C. Cells were incubated with serum-free medium (SS) for 12 h at 37°C and an atmosphere of 5% CO₂ and incubated with different concentrations of the CDP mix. After 12 h of treatment, the cells were washed with PBS. Cells were fixed with paraformaldehyde (PFA at 4%) for 10 min on ice. Then, cells were incubated with DAPI (1 : 1,000) for 10 min at room temperature. Finally, cells were washed with PBS, and the cover glass was removed and placed into a holder with a drop of PBS and glycerol 1 : 1. Cultured cells were photographed using an inverted phase-contrast microscope (Carl-Zeiss HB0-50, San Diego, CA, USA) equipped with an AxioCam/Cc1 digital camera. Cultures of HeLa and Caco-2 cells were grown in CM and incubated with DAPI and visualized using a confocal microscope (Olympus FV1000, Center Valley, PA, USA). The cells were observed by fluorescence emission between 405 and 505 nm.

At 28 days post-injury, the spinal tissues obtained from experimental mice were fixed in 4% paraformaldehyde (PFA) (4° C) in PBS (pH 7.4) for 20 min, embedded in paraffin, and sectioned at 4 μm thickness. Then, the apoptosis in the spinal tissues was detected by One Step TUNEL Apoptosis Assay Kit (Beyotime Biotech., Jiangsu, China), TUNEL positive cells were observed under fluorescence microscopy (HB050; Zeiss, Hamburg, Germany) (×200 magnification).

Brain tissue was postfixed in 4% paraformaldehyde, dehydrated with sucrose solution and sliced to 10 μm. Cultured neurons were fixed with 4% paraformaldehyde. Then they were permeabilized with 0.3% Triton X-100, and blocked with immunostaining blocking solution (Epizyme, Shanghai, China). After incubation with diluted primary antibody overnight at 4 ℃, the membranes were washed 3 times for 10 min with PBS and Tween 20. The antibodies were as follows: anti-PDK4 (1:200, 89295, Abcam), anti-NeuN (1:200, 26975-1-AP, Proteintech, Rosemont, IL, USA), anti-Iba1 (1:200, 5076, Abcam), anti-GFAP (1:200, 4648, Abcam). The next day, they were incubated with corresponding secondary antibodies: anti-rabbit Alex Fluor 488-conjugated secondary antibody (1:200, A11008, Invitrogen), anti-rabbit Alexa Fluor 594-conjugated secondary antibody (1:200, A32754, Invitrogen), anti-mice Alexa Fluor 594-conjugated secondary antibody (1:200, A32740, Invitrogen), anti-mice Alexa Fluor 488-conjugated secondary antibody (1:200, A32723, Invitrogen). Immunofluorescence images were captured by using the microscope (ZEISS, HB050, Berlin, Germany) and analyzed with ImageJ.

HeLa cells was seeded in 12-well flat-bottomed plates at a density of 1 × 10⁴ cells per well with 1 mL of CM and incubated for 24 h at 37 °C with 5% CO₂. Cells were incubated with serum-free medium (SS) for 12 h at 37 °C and an atmosphere of 5% CO₂ and incubated with different concentrations of the CDPs. After treatment, the cells were washed with PBS. Cells were fixed with paraformaldehyde (PFA at 4%) for 10 min on ice and collocated on cover glass, placed into a holder with a drop of PBS and glycerol 1:1 and photographed using an inverted phase-contrast microscope (Carl-Zeiss HB0-50, Gottingen, Germany) equipped with an AxioCam/Cc1 digital camera (Carl-Zeiss, Gottingen, Germany). Additionally cell cultures were observed directly using a confocal microscope (Olympus FV1000), images of the HeLa cells were taken using 40× magnification.

Immunofluorescence staining was performed following the procedures of our laboratory.^{39 (link)} Briefly, brain tissue was sliced (6 μm) and blocked with 5% normal fetal bovine serum in PBS containing 0.1% Triton X-100 for 1 h at room temperature prior to incubation with primary antibody overnight at 4 °C. After sections were washed three times with PBS for 45 min, they were incubated with proper secondary antibodies (Alexa Fluor 488 and Alexa Fluor 594, 1:200) for 2 h at room temperature. The slides were washed with PBS again three times for 45 min prior to be counterstained by 4,6-diamidino-2-phenylindole (DAPI) for 2 min. After three washes again, the slides were covered by microscopic glass with anti-fade mounting medium for further study. Negative controls were prepared by omitting the primary antibodies. Fluorescence microscopy imaging was performed using ZEISS HB050 inverted microscope system and handled by Image-Pro Plus 6.0 software (Media Cybernetics, Rockville, MD, USA) and Adobe Photoshop CS5 (Adobe Systems, San Jose, CA, USA).

Mice were perfused with PBS and 4% paraformaldehyde after anesthetization. Human brain tissue was postfixed in paraformaldehyde immediately after collection for immunofluorescence staining. Sections of 10 μm were collected from brains after being postfixed in paraformaldehyde for 24 h. Primary neurons seeded in 24-well plates were postfixed in paraformaldehyde for 30 min for immunofluorescence analysis. Cells were stained for TRAF3 (1:100, AB36988, Abcam), NeuN (1:200, ABN78, Millipore), Iba1 (1:200, AB5076, Abcam), GFAP (1:200, AB7260, Abcam) overnight at 4 °C. Primary antibodies were visualized using corresponding secondary antibodies (1:200, Millipore). Immunofluorescence images were captured using the microscope (ZEISS, HB050, Germany)^{22 (link)}.

Paraffin sections were deparaffinized and hydrated using the following incubation steps: 10 min in xylene twice; 5 min in 100%, 10 min in 95%, 10 min in 85%, and 10 min in 70% ethanol; and 5 min three times in PBS at room temperature. Antigen retrieval was achieved by boiling the sections in 10 mM sodium citrate for 10 min in a microwave oven. The sections were washed with PBS three times, and treated with 3% H₂O₂-methanol for 15 min. Immunostaining was performed by incubation with antibody against cleaved caspase 3 (1:200; Cell Signaling Technology, Beverly, MA, USA) for 2 h. Sections were then washed three times and incubated with secondary antibodies labeled with horseradish peroxidase for 30 min at room temperature. Immunohistochemical (IHC) imaging was performed using ZEISS HB050 (Zeiss, Jena, Germany) inverted microscope system and handled by Image-Pro Plus 6.1 software (Media Cybernetics, Rockville, MD, USA). Cells with brownish-yellow cytoplasm were counted as positive cells. The numbers of caspase 3 immunoreactive cells in the hippocampal CA1 and dentate gyrus (DG) regions were counted and analyzed in four microscopic fields (at ×200 magnification) by an investigator blinded to the treatment conditions.