Axiovs40

Cells were first fixed with 4% paraformaldehyde for 20 min at room temperature. Following the removal of the fixative by washing with PBS, cells were treated with cold methanol for 20 min at −20 °C for permeabilization. Five percent FBS (Thermo Fisher Scientific, Waltham, MA, USA) in PBS was applied for 2 h to block unspecific binding. Primary antibodies for MIST1 (Abcam, ab107390,1:100), rabbit polyclonal anti-TCF3 antibody (Santa Cruz, sc763, 1:100), goat polyclonal anti-cMyc antibody (Bethyl, A190-104A, 1:100), rabbit polyclonal anti-AMY1 antibody (Biomatik, CAU25843, 1:100), anti-rabbit polyclonal anti-M3R antibody (Abcam, ab126168, 1:100), rabbit polyclonal anti-AQP5 (Abcam, Ab78486, 1:100), and anti-rabbit polyclonal anti-CK19 antibody (Abcam, ab52625, 1:100) were diluted in 5% FBS blocking buffer and applied on cells overnight at 4 °C. An anti-rabbit/goat Alexa Fluor™ 568 or 488 nm conjugated antibodies (1:200) was used as a secondary antibody for 2 h at room temperature followed by a final wash. Endoplasmic reticulum staining was performed by an ER-GFP, BacMam 2.0 labeling kit (Cell light, C10590). Images were obtained using a Zeiss Axiovert 200M microscope equipped with a AxioCam MRm camera and AxioVs40 software (Ver. 4.7.1.0) (Zeiss, Oberkochen, Germany).

The in vitro cell migration assays were performed by using a modified Boyden chamber inserted with polyethylene terephthalate filter membrane containing 8 µm pores in 24-well plates (Millipore, Milan, Italy). The upper chamber contained C6 glioma cells or HMEC-1 cells in serum-free culture medium, and the lower chamber contained TCM or ICM. After 24 h of incubation, migrated cells were fixed in 100% methanol for 1 h and then stained with Giemsa stain (Sigma) for 10 min and images were processed using AxioVs40 software (Ver. 4.8.0.0, Carl Zeiss, Germany). Data were obtained by measuring ten randomly selected fields of transwell-migrated cells using Image-Pro Plus 6.0 software (Media Cybernetics, Silver Spring, MD, USA).

Co-cultured or control mMSCs grown on coverslips (BD BioCoat™) were fixed with 4% paraformaldehyde for 20 minutes. After fixation, the cells were washed, permeabilized, and blocked in phosphate-buffered saline (PBS) containing 0.2% Triton X-100 and 5% FBS for 30 minutes at room temperature. Cells were then incubated with goat polyclonal anti-Ptf1α (1: 200), rabbit polyclonal anti-Mist-1 (1: 200) in PBS containing 0.1% Triton X-100 and 1% FBS at 4°C overnight. After washing with PBS, the cells were incubated at room temperature for 1 h with appropriate fluorescence-conjugated secondary antibodies (1: 200 dilution, Molecular Probes). Coverslips were mounted and nuclei stained with Vectashield mounting solution containing DAPI (Vector Laboratories Ltd). Fluorescence was observed under a 100X magnification using a Zeiss Axiovert 200M microscope equipped with a Zeiss AxioCam MRm camera and images were obtained from AxioVs40 software (Ver. 4.7.1.0, Zeiss).

Alkaline phosphatase blue in situ hybridization stained embryos were mounted for brightfield microscopy in 80% glycerol, 20% PBST, 1 mM EDTA. Images were obtained using an AxioCam CC1 on an AxioPlan2 microscope with a PLAN-NEOFLUAR 20×/0.5 or 10×/0.3 objective and DIC optics using the AxioVs40 Software (all Zeiss). From image stacks (1 μm step size), minimum intensity projections of z-planes as indicated in the figure legends were generated using ImageJ. Embryos stained by fluorescent in situ hybridization and immunohistochemistry were recorded using a Zeiss LSM 510 or LSM 880. All figures were assembled using Adobe Photoshop CS4 or CS6. When linear adjustment of levels was made, histograms were clipped to the same values for experimental and control images.