Axio observer z1

To coat samples with LN, PVA, PVA-G, and PVA-F were incubated in 500 μl of 20 μg ml⁻¹ LN solution at 37 °C for 1 h and washed with sterile 1× PBS once for 5 min before cell seeding. To compare the amount of LN on LN-coated PVA, PVA-G, and PVA-F, 5% of Alexa 488 fluorescent-labeled LN was mixed with non-fluorescent LN. The LN-coated samples were imaged with Zeiss fluorescence microscope (Axio Observer Z1), and the fluorescence intensity was analyzed using Fiji software.
To bind NGF, sterile PVA, PVA-G, and PVA-F were incubated in 100 μl of 100 μg ml⁻¹ recombinant human beta-NGF (β-NGF, PeproTech, Inc.) solution at 37 °C for 1 h and used directly for cell seeding. To compare the coated NGF on PVA, PVA-G, and PVA-F, the NGF-coated samples were stained with NGF antibody (PeproTech, Inc.) and imaged with Zeiss fluorescence microscope (Axio Observer Z1). The fluorescence intensity was analyzed using Fiji software.

Striatal GFP neurons were examined on sections using a Zeiss Axio Observer Z1 fluorescent microscope equipped with a Zeiss Colibri LED illumination system and a Zeiss AxioCam MRc camera (Zeiss, Oberkochen, Germany), with a 20× objective. We used 9 × 9 tiles with a 10% overlap to create panoramic images. Mosaic stitching was carried out using AxioVision 40 V 4.8.2.0 software (Zeiss, Oberkochen, Germany).
The diameter and area of the neuron cell bodies were determined on sections with the AxioVision 40 V 4.8.2.0 software using the line and outline tools, respectively. This analysis was performed on cells with a visible nucleus and axonal hillock. The neuron size was determined by drawing a line from the axonal hillock to the opposite edge of the cell.
For microscopy of living neurons stained with DRAQ5 and GFP, the sorted suspension was placed on a coverslip and covered with a round coverslip, 15 mm in diameter. The resulting preparations were studied under a Zeiss Axio Observer Z1 fluorescent microscope. Images were acquired at a 40× objective and analyzed using AxioVision 40 V 4.8.2.0 software. Living neuron microscopy was performed over the entire area of the coverslip, 176.7 mm², analyzing images of all sorted cells.

MDA-MB-231 cells were seeded in a 96-well plate (15∙10³ cells per well), grown overnight and then cultured with 50 μM of various LC-PUFAs for 24 h.
For lipid droplets staining by Oil Red O, cells were washed twice with PBS, fixed with 10% formalin for 45 min, and then rinsed twice in water for 1 min, followed by 5 min in 60% isopropanol. The cells were stained with Oil Red O (1.8 mg/mL in 60% isopropanol) for 15 min and rinsed 5 times with ddH₂O to remove excess stain. Oil Red O stained cells were directly visualized and imaged using an inverted fluorescent microscope Axio Observer Z1 (Carl Zeiss). Quantification of lipid accumulation was achieved by Oil Red O extraction with 100% isopropanol and gentle agitation for 5 min at room temperature. Then the extracts were transferred to a new 96-well plate and absorbance was measured at 492 nm using X-mark plate reader (BioRad).
For lipid droplet staining with BODIPY, cells were washed twice with DPBS, fixed with 10% formalin for 45 min and then washed twice with DPBS for 1 min. Cells were incubated with 2 μM BOBIPY (Lumiprobe) in the dark at 37°C for 60 min and then washed 3 times with DPBS to remove excess dye. Cells stained with BOBIPY were visualized directly with an inverted fluorescent microscope Axio Observer Z1 (Carl Zeiss).

Imaging was done using an inverted microscope (IX71, Olympus, Japan) equipped with a CCD camera (DP72, Olympus, Japan) and a mercury lamp (USH-1030L, Olympus) or on an Axio Observer Z1 (Zeiss, Germany) inverted microscope using an EC Plan-Neofluar 20× objective and equipped with a Hamamatsu CMOS camera (Hamamatsu, Japan). Illumination for the Axio Observer Z1 was provided by a Colibri 2 LED Illumination System with a 120 V LED illuminator (Zeiss, Germany). Rhodamine-DPPE was illuminated using a red filter.