Axio observer 7 0 apotome 2

All the images were acquired using 63× oil immersion lens in Axio Observer 7.0 Apotome 2.0 (Zeiss) microscope. The 3D Z-stack images were obtained with optimum slice size of 0.24 μm. The slice size may vary depending on cell thickness. After acquiring the 3D Z-stacks images were processed for orthogonal projections to demonstrate the cross-sections of the cells. The quantitative analysis of the fluorescence intensities were done for the respective orthogonal projections by ZEN image processing software and the mean intensities were plotted using Sigma Plot 10.2.

Neuro2a cells were maintained in Advanced DMEM supplemented with 10% FBS and antibiotics penicillin-streptomycin. For immunofluorescence microscopy studies 5 × 10⁴ cells were seeded on glass coverslip in a 12 well culture plate for 24 hours. Four experimental groups were maintained as cell control, EGCG (100 μM) treated, MG (1 mM) treated and MG+EGCG treated. Treatment was carried out for 24 hours after which, the cells were fixed with ice-cold methanol. Cell permeabilization was carried with 0.2% TritonX 100. After 3 PBS washes cells were blocked in 5% horse serum for 1 hour at 37°C. Cells were incubated with primary antibodies at 4°C overnight. Further, cells were washed with 1X PBS and incubated with respective antibodies anti-mouse secondary antibody conjugated with Alexa flour-488, Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody with Alexa Fluor 555 (A-21428) followed by 300 nM DAPI. The coverslips were mounted in 80% glycerol and sealed and were observed under 63× oil immersion lens in Axio Observer 7.0 Apotome 2.0 (Zeiss) microscope using ZEN pro software.