Sp5 2 microscope

Plastic-embedded samples (WT = 3, RAG1^−/− = 4, D7) were used to visualize collagen fibrils in WT and RAG1^−/− mice via SHG imaging. Collagen fibrils exhibit endogenous SHG signals arising from their well-known non-centrosymmetric molecular structure (20 (link), 21 (link)). Imaging was performed using a Leica SP5 II microscope (Leica Microsystems, Wetzlar, Germany). The SHG signal was generated using a Mai Tai^® HP Ti:Sapphire oscillator (Spectra Physics, Stahnsdorf, Germany) with 100 fs pulse width at 80 MHz and wavelength of 910 nm. The SHG collagen signal was detected in the range of 450–460 nm. Z-Stacks were recorded with 4 µm z-spacing using 25× water immersion objectives with numerical apertures of 0.95. Overview images were created via image stitching and maximal intensity projections of z-stacks.

Indirect immunofluorescence against HLA-G in WJ-MSCs obtained from non-vitrified (n = 5) and vitrified (n = 5) WJ tissue samples was performed. An average of 1 × 10⁴ WJ-MSCs were seeded on culture slides (Sigma-Aldrich, Darmstadt, Germany), followed by addition of 1 mL of standard culture medium. After 10 days, the culture slides were microscopically checked and when confluency observed, the indirect immunofluorescence was performed. For this purpose, the WJ-MSCs were fixed for 10 min with 10% v/v neutral formalin buffer (Sigma-Aldrich, Darmstadt, Germany). Antigen retrieval and blocking of cells was applied in all samples, followed by addition of monoclonal antibody against human HLA-G (1:1000, Catalog MA1-10359, ThermoFisher Scientific, Waltham, MA, USA). Secondary FITC-conjugated mouse IgG antibody (1:100, Sigma-Aldrich, Darmstadt, Germany) was added. Cell nuclei became evident with DAPI staining (ThermoFisher Scientific, Waltham, MA, USA). Finally, the slides were glycerol mounted and observed under fluorescent microscope. Images were acquired with LEICA SP5 II microscope equipped with LAS Suite v2 software (Leica, Microsystems, Wetzlar, Germany).

pEGFP-TI-VAMP, pEGFP-VAMP3 and pEGFP-Rab11 (Thierry Galli, University Pierre and Marie Curie, Paris, France); pEGFP-Rab5 (kind gift from Letizia Lanzetti, University of Turin, Turin, Italy); pEGFP-CD63 (kind gift from John Paul Luzio, Cambridge University, Cambridge, UK).
Plasmids (1 μg ml⁻¹) were transfected in cultures of astrocytes using Lipofectamine 2 000 (3 μg ml⁻¹; Invitrogen) according to the manufacturer’s instructions. At 48 h after transfection, astrocytes were treated with Plg⁶⁴⁷ (25 nm) or Pln⁴⁸⁸ (25 nm) during 2 h and live cells were imaged using a Leica SP5 II microscope.

DLD-1 human colon carcinoma cells were maintained as monolayers in low glucose DMEM supplemented with 10% FBS. The cells were incubated in a humidified environment at 37°C with 5% (v/v) CO₂, and sub-cultured using trypsin to detach cells.
Confocal imaging of monolayer and spheroid tumour models were performed on a Leica SP5 II microscope, and images were analysed using LAS AF Lite. A heated stage was used to maintain the temperature at 37°C during live cell imaging. The samples were excited with 488 nm light, and the emission wavelengths collected between 530–650 nm.