Phase contrast light microscope

To assess calcium deposition, Alizarin Red S staining was carried out on DPSC cultures. On days 7, 14, 21, and 28, the cell supernatant was discarded and cells were washed twice with PBS with Ca/Mg. After that, cultures were fixed with 4% paraformaldehyde (PFA) for 30 minutes and then washed with PBS. Samples were afterwards incubated with Alizarin Red S (40 mM in deionized water) for 30 minutes in the dark. After that, DPSCs were washed three times with deionized water and samples were observed under a phase-contrast light microscope (Leica, Wetzlar, Germany) equipped with a CoolSNAP camera to acquire computerized images (Photometrics, Tucson, AZ).

Tumor tissues were fixed with 10% formalin overnight and then embedded with paraffin. The tissue was cut into 4 μm sections, and the sections were dewaxed for immunohistochemistry. H&E staining was performed using a commercial staining kit (ab245880, Abcam) according to the manufacturer's instructions. For IHC staining, the section was blocked with 5% BSA for 1 h and then incubated with anti-Ki-67 antibodies (1 : 1000, ab270650, abcam) overnight. After washing with TBST buffer, the section was soaked with 2 drops of SignalStain® Boost Detection Reagent (HRP, Rabbit #8114, Cell Signaling Technologies) for 30 min at room temperature. Signal development was performed for 5 min using SignalStain® Substrate (#8059, Cell Signaling Technologies). The images were captured using a phase-contrast light microscope (Leica, Germany).

Tissues were fixed with 4% paraformaldehyde for 48 h, and 3-μm thick tissue sections were prepared. Sections were subsequently deparaffinized and rehydrated using conventional methods. Antigen retrieval was performed with Antigen Unmasking Solution (Vector Laboratories, United States) at 95°C for 10 min. Sections were immersed in 3% hydrogen peroxide in methanol for 15 min to quench endogenous peroxidase activity and were blocked with 10% normal goat serum for 30 min at room temperature. Sections were incubated with primary antibodies against Runx2 (ab76956; 1:200, Abcam, United Kingdom), Osteopontin (OPN; ab69498; 1:200, Abcam, United Kingdom), and osteocalcin (OCN; ab93876; 1:400, Abcam, United Kingdom) at 4°C overnight. Then, sections were incubated with the corresponding biotinylated secondary antibody (Vector Laboratories, United States) for 30 min at room temperature prior to reaction with DAB chromogen (Vector Laboratories, United States). Sections were counterstained with hematoxylin, and micrographs were acquired with a phase contrast light microscope (Leica, Germany).

The morphology of the NPC-collagen microspheres before and after decellularization, the NPC-derived ECM following repopulation with hDFs, and the hDF-collagen microspheres were observed via a Leica phase-contrast light microscope (Leica DMIL, Solms, Germany) and images were captured with a Nikon COOLPIX5400 camera (Nikon, Tokyo, Japan). The viability of the NPCs in the microspheres before and after decellularization, the hDFs seeded in the NPC-derived decellularized matrix and the hDFs seeded in the collagen microspheres was determined by live/dead staining at 6 and 18 days. Cells were incubated for 45 min in the dark with 2 μM calcein acetoxymethylester and 4 μM ethidium homodimer-1 (LIVE/DEAD Viability/Cytotoxicity Kit; Molecular Probes, Invitrogen, Carlsbad, CA, USA), to stain the live and dead cells, respectively. The microspheres were then washed with PBS and examined under a Nikon inverted fluorescent microscope Eclipse TE2000-U equipped with a SPOT FLEX camera (Diagnostic Instruments, Sterling Heights, MI, US).

HGFs were grown on 24-well culture plates and allowed to adhere. After 24 h, the medium was replaced with 1 ml of DMEM 1% sucrose, then each thermoset (ø = 14 mm; h = 2.5 mm) was cut into four equal parts and one part was placed in each well on the cells. When called for, standardized bacterial culture was added. After 24 and 48 h, the observation was carried out using a phase-contrast light microscope (LEICA, Wetzlar, Germany) equipped with a CoolSNAP videocamera for acquiring computerized images (Photometrics, Tucson, AZ).

After I/R treatment, some rat brains were fixed in 10% neutral buffered formalin (NBF) and embedded in paraffin. Paraffin sections were stained with Hematoxylin and Eosin (H&E) or terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling (TUNEL; Roche, 11966006001, Germany) or antibody against Ctgf (Santa Cruz Biotechnology, sc-101586, U.S.A.). The images of H&E and immunohistochemistry were measured through phase-contrast light microscope (Leica, Wetzlar, Germany), while the images of TUNEL were examined under the inverted fluorescence microscope (Leica). For TUNEL assay, the rate of apoptotic events in total cells was calculated.

Obtained during surgery, fresh tumor and thrombus tissues were stored in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) with 10% fetal bovine serum (FBS, Gibco) and processed on ice. Subsequently, the tissues were rinsed with cold phosphate buffer saline (PBS, Gibco) three times and minced into ~1mm³ pieces. The tissue pieces were digested into single-cell suspensions using collagenase-2 (1 mg/mL) at 37 °C for 45 min and filtered through a 70-μm cell strainer. Upon digestion, the single-cell suspensions were centrifugated at 400 g for 5 min and the supernatant was discarded. Adding red blood cell lysis buffer (Solarbio) to remove red blood cells, the suspensions were then passed through a 40-μm filter and centrifugated at 500 g for 5 min, and resuspended in PBS. Cell viability was validated under the phase-contrast light microscope (Leica) after staining with trypan blue (ACMEC).