Eclipse ts2r fl inverted microscope

10 µL of the ZapB-GFP, ZapB-mCherry and Aβ42-GFP IB samples in buffer A (50 mM Tris, 100 mM NaCl, pH 7.4) were deposited on top of microscopy glass slides and covered with coverslips. The fluorescence was observed using an Eclipse Ts2R-FL inverted microscope (Nikon, Japan) using a C-LED470 filter for GFP fluorescence and a C-LED525 for mCherry fluorescence.
To determine the GFP-fluorescence intensity of ZapB-GFP and Aβ42-GFP IBs, images were analyzed using the ImageJ software. 50 fluorescent dots were selected in each image maintaining the same dimensions (height × width) for the different selections. After that, the intensity of these different fluorescent dots was calculated and the average and SEM values were estimated.

Cells and tissues were stained to detect free cholesterol using commercially available Filipin III (Sigma-Aldrich), according to the manufacturer's instructions. Slides were fixed with 4% paraformaldehyde for 30 min, washed with PBS, and incubated with freshly prepared filipin solution (100 μg/mL) for 1 h in the dark. The slides were then washed with PBS, and a drop of glycerol in PBS (glycerol: PBS = 1:9) was added. The samples were visualized using an ECLIPSE Ts2R-FL inverted microscope (Nikon, Tokyo, Japan).

For estimation of cell migration with Boyden’s chambers, cultures of SKOV-3 cells at 75–85% of confluence were fasted (8 h), then 5 × 10⁴ cells were placed in the upper compartment of Boyden´s chamber insert (Millicell-Millipore, Sigma-Aldrich, St. Louis, MO, USA) with the indicated pharmacological treatments diluted in serum and antibiotic free-RPMI medium. In the lower compartment, RPMI complemented with a 10% fetal bovine serum was placed as a chemoattractant. Chambers were incubated for 16 h. After this, the inserts were washed with a phosphate buffer (PBS, in mM: 136 NaCl, 2.7 KCl, 10 Na₂HPO₄, 1.8 KH₂PO₄, pH 7.4), fixed for 20 min in a 4% paraformaldehyde diluted in PBS and stained with a 0.5% crystal violet. Pictures were acquired with a Nikon eclipse Ts2R-FL inverted microscope. A total of 6 pictures of 3 independent experiments were counted using the ImageJ plugin Cell Counter (NCBI-USA).