Lsm710 laser scanning spectral confocal microscope

Astrocytes and SH-SY5Y cells were grown on 25 mm coverslips to carry out the corresponding treatments the day of the ΔΨm acquisition, cells were incubated with 40 nM tetramethyl-rhodamine methyl ester (TMRM) in a HEPES-buffered salt solution (HBSS; 156 mM NaCl, 3 mM KCl, 2 mM MgSO₄, 1.25 mM KH2PO₄, 2 mM CaCl₂, 10 mM glucose and 10 mM HEPES; pH = 7.35) for 40 min using the Attofluor Cell Chambers (Thermo Fisher). The dye was present in the chamber at the time of image acquisition. TMRM, a lipophilic fluorescent dye, is accumulated in active mitochondria and its fluorescence intensity is directly proportional to ΔΨm [112 (link)]. Therefore, a reduction in TMRM fluorescence represents mitochondrial depolarization. Confocal images were obtained using a LSM710 laser scanning spectral confocal microscope (Zeiss, Oberkochen, Germany) equipped with a META detection system and a ×40 water immersion objective. TMRM was excited using the 560 nm laser line and fluorescence was measured above 580 nm. The Z-stack images were analyzed using the Volocity Quantitation software 6.5.1 (Quorum Technologies, Puslinch, ON, CANADA) and TMRM values for control cases were set to 100%.

The different cell types were seeded in 24-well plates containing poly-D-lysine (20 µg/mL) pretreated glass coverslips. After treatments, they were fixed for 20 min with 4% paraformaldehyde and blocked for 30 min at 37 °C with 5% goat serum in a solution of 1% Triton X-100 in PB. After that, cells were incubated for 1 h at 37 °C with different primary antibodies: mouse anti-C/EBPβ (C19 clone, Santa Cruz, ref. sc7962), rabbit anti-TFAM (Cell Signaling, Leiden, The Netherlands, ref. 8076), or rabbit anti-p62 (Sigma, ref. P00067). Cover-slips were then washed with PB and incubated for 45 min at 37 °C with Alexa-labeled secondary antibodies (Thermo Fisher, Madrid, Spain). Staining of nuclei was performed using 4′,6-Diamidino-2-phenylindole (DAPI). Images were acquired in a LSM710 laser scanning spectral confocal microscope (Zeiss) and the microscope settings were adjusted to produce the optimum signal-to-noise ratio. Quantitative analysis of fluorescence intensity and particles number was performed by the use of Image-J software, and at least five different counting fields were randomly selected from three independent experiments.

Cells were processed for immunocytochemistry as previously described^{69 (link)}. Briefly, NS grown on glass were previously fixed 15 minutes at room temperature in 4% paraformaldehyde and incubated at 37 °C for 1 h with primary antibodies directed against ki67 (1/200, rabbit, Abcam), β-III-tubulin (1/400, TuJ-1 clone; rabbit; Abcam), MAP-2 (1/200, mouse; Sigma), CNPase (rabbit, Covance) and GFAP (1/500, mouse; Sigma). After several rinses in PBS, samples were then incubated with Alexa-488 goat anti-rabbit and Alexa-647 goat anti-mouse antibodies (1/500, Molecular Probes) for 45 min at 37 °C. Staining of nuclei was performed using 4′,6-diamidino-2-phenylindole (DAPI, 1/500). Finally images were acquired in a LSM710 laser scanning spectral confocal microscope (Zeiss). Confocal microscope settings were adjusted to produce the optimum signal-to-noise ratio.