Fv1000mpe two photon microscope

Samples were fixed with 4% paraformaldehyde for 30 min; blocked with 5% normal goat serum (NGS); and permeabilized with 0.1% triton-x (Bio-Rad, München, Germany) for 1 h. Cells were stained with anti-fibronectin antibody (clone A-11; Santa Cruz Biotechnology, Dallas, USA); actin was stained using Alexa-488 conjugated phalloidin (BioMol, Hamburg, Germany); and nuclei were counterstained with DAPI (10 ng/ml) for 10 min. Samples were analyzed with a FV1000MPE two-photon microscope to investigate 3D growth (Olympus Corp., Tokyo, Japan). To analyze viability of iPSC colonies in fibrin gel, we stained living cells with Calcein-AM and dead cells with propidium iodide (PI) (both Sigma Aldrich).

Brain slice imaging was performed as described previously^{25 (link)}. In brief, the animals were anesthetized with Avertin, and acute brain slices containing the hippocampus region were prepared in cold slicing buffer containing: 110 mM choline-Cl, 2.5 mM KCl, 1.25 mM NaH₂PO₄, 25 mM NaHCO₃, 7 mM MgCl₂, 25 mM glucose, and 2 mM CaCl₂. Slices were allowed to recover at 35 °C in oxygenated Ringers solution containing: 125 mM NaCl, 2.5 mM KCl, 1.25 mM NaH₂PO₄, 25 mM NaHCO₃, 1.3 mM MgCl₂, 25 mM glucose, and 2 mM CaCl₂ for at least 40 min before experiments. An Olympus FV1000MPE two-photon microscope equipped with a ×40/0.80 NA water-immersion objective and a mode-locked Mai Tai Ti:Sapphire laser (Spectra-Physics) were used for imaging the slices. The two-photon excitation wavelength was set as 880 nm, except where indicated otherwise. Formaldehyde or acetaldehyde were added to the physiological solution and perfused into the chamber with slices during imaging. Imaging files were processed with ImageJ 1.52a (NIH) to measure fluorescence intensity and to generate the ratiometric pseudo-color images. Intensity traces were generated by Origin 2018 and pseudo-color heatmap images were generated by custom-written MATLAB programs.

On day 21 after implantation of LN229-RFP-luc/U87-eGFP, LN229-RFP-luc/U87-C and LN229-RFP-luc/U87-vC, mice (n = 4) were sacrificed and perfused transcardially with cold PBS and 4% PFA in PBS. The brains were then post-fixed in 4% PFA, dehydrated successively in 20% and 30% sucrose, embedded in optimal cutting temperature (OCT) compound (Sakura; Tokyo, Japan), frozen at -80 ℃ and sliced into coronal sections (8 μm and 80 μm). For 3D Z-stack imaging, 80 μm-thick sections were viewed using an Olympus FV-1000MPE two-photon microscope (Olympus; Japan) equipped with a water-immersion objective (XLPlan N 25×/0.05 W MP). The 80 μm-deep stacks were acquired with a 1.6 μm step depth and analyzed using Olympus FV31S-SW.
Next, 8 μm frozen sections or cells grown on glass coverslips were fixed with 4% PFA for 30 min, permeabilized with 0.2% Triton X-100 in PBS, blocked with 5% BSA for 1 h and incubated with primary antibodies against Caveolin1 (MAB5736; R&D), Caveolin2 (410700; Life technologies), EEA1 (66218-1-Ig; Proteintech), and Cavin1 (18892-1-AP; Proteintech) for 12 h at 4 ℃. Sections were then incubated with Alexa Fluor 488-, or 594-conjugated secondary antibodies (Life Technologies) for 1 h, followed by counterstaining with DAPI (C0060; Solarbio; Beijing, China). Images were captured using a confocal fluorescence microscope (Olympus, FluoView 1200; Tokyo, Japan).