Lsm510 axiovert 200m microscope

Mice were anesthetized and transcardially perfused with 4% PFA (w/v) in PBS (pH 7.4). Brains were removed, post-fixed for 2 hr in 4% PFA, cryo-protected in 30% (w/v) sucrose at 4°C and embedded in Tissue-Tek medium (Sakura Finetek Europe, The Netherlands). Free-floating cryosections (40 µm) were washed three times in Tris-Buffered Saline (TBS), permeabilized in 0.3% (v/v) Triton-X-100 in TBS for 5 min and incubated with blocking solution (10% (v/v) normal serum, 1% (w/v) BSA, 0.3% Triton-X-100 in TBS) for 2 hr. Sections were incubated with primary antibodies overnight at 4°C. After three washes in 0.025% Triton-X-100 in TBS sections were incubated with corresponding secondary antibodies. Sections were counterstained with Hoechst 33258 (H-3569, Molecular Probes, Grand Island, NY) for 5 min or with RedDot2 (40061, Biotium, Fremont, CA) for 20 min. After three washes in TBS sections were mounted with Roti-Mount FluorCare mounting medium (Carl Roth, Germany). Images were acquired using a LSM 510/Axiovert200M microscope (Carl Zeiss, Germany).
For cresyl violet staining free-floating brain sections (40 µm) were mounted on glass slides and incubated in cresyl violet (0.1 M sodium acetate, 2% acetic acid, 0.02 M cresyl violet acetate, Sigma, St. Louis, MO, in distilled water, pH 3.5).

To evaluate whether MSCs were responsive to insulin, glucose uptake and the cellular localization of GLUT4 were first evaluated in MSCs not treated with GCs (Exp 1 and 2) from T1 to T6.
For the glucose uptake assay, 3x10³ cells/well were plated in a 96-well plate and treated according to the above protocol; after insulin stimulation, 10 mM of 2-deoxyglucose (2-DG) was added for 20 minutes, and a colorimetric assay was performed following the manufacturer’s instructions. The readings were at 420 nm in a microplate reader (Thermo Scientific Multiskan GO Microplate Spectrophotometer, Milano, Italy).
For the cellular distribution of GLUT4, 1.5x10⁴ cells (Exp 1 and 2 derived from the 7 patients) were seeded in triplicate on coverslips and treated as indicated before until T5 sampling time. Cells were then washed, fixed with 4% PFA and permeabilized for 30 min. Subsequently, cells were incubated with anti-GLUT4 antibody (Santa Cruz Biotechnology, USA) followed by treatment for 30 min with a goat anti-mouse FITC-conjugated antibody (23 (link)). Finally, coverslips were mounted on glass slides in Vectashield (Vectorlabs, CA, USA), and confocal imaging was performed using a Zeiss LSM510/Axiovert 200 M microscope with an objective lens at 20× magnification (24 (link)). Line scans were acquired excluding nuclear regions, and GLUT4 immunofluorescence was analyzed as described elsewhere.