Tecnai biotwin tem

Aux-KO/SJ1-KI^RQ mice were transcardially perfused with 4% formaldehyde and 0.125% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4). Dorsal striata were dissected and embedded in 1% gelatin in 0.1 M phosphate buffer. Tissue pieces were trimmed, infiltrated in 2.3 M sucrose, and then were frozen rapidly onto aluminum pins in liquid nitrogen. 60 nm frozen sections on carbon/formvar coated grids were prepared with a Leica Cryo-EMUC6 UltraCut microtome. Sections were labeled with rabbit anti-Tyrosine Hydroxylase (TH) and 10 nm Protein A gold (Utrecht Medical Center)^{44 (link)}. Grids were examined in FEI Tecnai Biotwin TEM at 80Kv. Images were taken with Morada CCD and iTEM (Olympus) software.

For conventional Epon embedding, cells were fixed in 2.5% glutaraldehyde/2% sucrose in 0.1 M sodium cacodylate buffer pH 7.4 (NaCaCo buffer) for 30 min at RT followed by 30 min at 4 °C. Subsequently, cells were rinsed with NaCaCo buffer and further processed as described38 (link).
Samples were viewed using an FEI Tecnai Biotwin TEM at 80 Kv. Images were collected using Morada CCD and iTEM (Olympus) software.
Epon-embedded EM samples were first inspected to qualitatively determine whether the respective Mel220 transfectant formed fibril-containing melanosomes. To quantify fibril formation, we then counted fibril-containing organelles in 15 arbitrarily chosen cells in one view field. Each count was performed once and the respective means are indicated in Figs 3A and 4D,E,I,J, and Suppl. Fig. S7. A Student’s two-tailed t-test (Y151F and F207L/215 L) or a one-way ANOVA with Dunnett’s post test (all others) was used to determine whether means are statistically different from the wt-PMEL sample. Asterisks in respective figures indicate statistical significance, but are shown in brackets for construct G165A where only Dunnett’s post test but not the one-way ANOVA indicated statistical significance.

Transmission electron microscope (TEM) was used to investigate the morphology of the EVs isolated by ultracentrifugation. Briefly, EVs at an optimal concentration were first placed on 400 mesh carbon/formvar-coated grids and allowed to be absorbed on formvar for a minimum of 10 min. Next, the grids (membrane side down) were transferred to a 50-μl drop of 2.5% glutaraldehyde for 5 min, after which they were transferred to a 100-μl drop of distilled water and were left to stand for 2 min. This process was repeated nine times for a total of 10 water washes. Then, the sample was loaded on the grid and stained by 4% uranyl acetate for 10 min and 1% methylcellulose for 5 min. The remaining water was removed using filter paper. Finally, the samples were viewed using a Tecnai Bio Twin TEM (FEI, Hillsboro, OR, USA), and images were obtained using an AMT CCD camera (Advanced Microscopy Techniques, Woburn, MA, USA).

Transmission electron microscopy (TEM) analysis was performed to examine the morphology of the bioglass nanoparticles with and without the chitosan coating. The nanoparticles were suspended in water, then picked up on Cu grids under the optical microscope and imaged on a Tecnai BioTWIN TEM (FEI, Hillsboro, OR, USA) at an accelerating voltage of 80 kV. Then, 3D projections of the TEM images were obtained with the use of the Gwyddion 2.45 freeware.