Axiovert 40 inverted microscope

A Matrigel-based tube formation assay was used to assess the inhibitory effect against VMs. Briefly, 200 μL of Matrigel (BD Biosciences, Franklin Lakes, NJ) was plated onto 24-well cell plates, and incubated for 30 min until solidification. C6 cells (1 × 10⁴) were resuspended with serum-free medium containing free DOX, DOX-HA-LPs, and free HA, and seeded onto the Matrigel. The final concentration of DOX was 5 μg/mL. The content of HA in free HA was the same with that in DOX-HA-LPs. After incubation at 37 °C for 1 h and 4 h, the state of VMs was analyzed by Carl Zeiss Jena Axiovert 40 inverted microscope (Oberkochen, Germany).

The in vitro cell migration and invasion assays were performed in 24-well plates (Corning, Inc., New York, USA) using modified Boyden chamber inserts with a polycarbonate filter membrane containing 8-μm pores. For the invasion assay, matrigel (BD Biosciences) was diluted 1:1 with serum-free medium and used to coat the filter membranes. The cells (1×10⁵) were suspended in 250 μl of serum-free DMEM and seeded onto the upper compartment of the transwell chamber; DMEM containing 10% FBS was used in the lower chamber for stimulation. After a 24 h or 72 h incubation for the migration or invasion analysis, respectively, the medium in the upper chamber was removed, and the filters were fixed in 10% formalin for 15 min. The cells on the lower surface were stained with 4′, 6-diamidino-2-phenylindole (DAPI; Sigma). Five fields were photographed at ×200 original magnification using a Zeiss Axiovert 40 inverted microscope and processed using the AxioVision Rel. 4.8.2 software. The cells were counted using the ImageJ program [43 (link)]. The migration and invasion data are reported as the number of cells per microscopic field. Five fields were analyzed.

Immunofluorescence was performed with primary antibodies against E-cadherin (24E10; #3195; Cell Signaling), pan-cytokeratin (pan-CTKR; ab6401; Abcam), cofilin (#3312; Cell Signaling), F-actin (NH3; MA1-80729; Thermo-Scientific), β-actin (C4: sc-47778; Santa Cruz), and vimentin (Clone V9; V6389; Sigma). Cells were incubated with either an anti-rabbit antibody conjugated with Alexa 488 or an anti-mouse antibody conjugated with Alexa 594 (Invitrogen). The nuclei were stained with DAPI. The digital images were obtained using a Zeiss Axiovert 40 inverted microscope and processed using the AxioVision Rel. 4.8.2 software (Carl Zeiss).

Cell spheres were cultured under standard differentiating conditions (DMEM/F12 supplemented with 10% FBS) and in Ultra Low Attachment plates (Corning). After 14 days in culture, cell morphology was assessed using a Zeiss Axiovert 40 inverted microscope with Axio-Vision software. Cell surface markers (CD44 and CD117) were detected by immunofluorescence staining. An epithelium differentiation marker, Cytokeratin-7 (CK-7), and ovarian cancer antigen-125 (CA125) were used for immunofluorescence staining, followed by incubation with a FITC-labeled goat anti-mouse or anti-rabbit IgG. Nuclei were counterstained with 4, 6-diamidino-2-phenylindole (DAPI; Santa Cruz).

Samples at day 21 were fixed in 4% neutral buffered formalin, washed with 2-propanol, and stained with an Oil Red O working solution (0.5% Oil Red O in 2-propanol, 60% in H₂O). After a further wash in 2-propanol, the cultures were washed and maintained in H₂O for imaging. Pictures were acquired using an Axiovert 40 inverted microscope, CP-Achromat 5×/0.12 objective, equipped with an Axiocam 105 color camera (Carl Zeiss Microscopy GmbH, Jena, Germany). At least 10 representative pictures were taken for further image analysis. Images were analyzed with Fiji/ImageJ software (NIH, Bethesda, MD, USA). Briefly, raw images were imported and converted to RGB. A color threshold was applied to select stained cells, then the area covered by the selection was measured (in µm²). The percentage out of the total area was calculated. Final results were calculated as the average % area of the 10 images.