Eclipse te300 c2 lscm

Fluorescence images were obtained using a commercial confocal microscope (Nikon Eclipse TE300 C2 LSCM, Nikon, Japan) equipped with a Nikon 60 × or 100 × immersion oil objective (Apo Plan, NA 1.4), and a custom-made two-photon fluorescence microscope (TPFM). Briefly, a mode-locked Ti: Sapphire laser (Chameleon, 120 fs pulse width, 90 MHz repetition rate, Coherent, CA) operating at 800 nm was coupled into a custom-made scanning system based on a pair of galvanometric mirrors (LSKGG4/M, Thorlabs, USA). The laser was focused onto the specimen by a refractive index tunable 25 × objective lens (LD LCI Plan-Apochromat 25X/0.8 Imm Corr DIC M27, Zeiss, Germany). The field of view was 450 × 450 μm², the resolution employed was 0.44 × 0.44 μm² or 1.75 × 1.75 μm² for high- and low-resolution reconstruction, respectively. The system was equipped with a closed-loop XY stage (U-780 PILine XY Stage System, Physik Instrumente, Germany) for the radial displacement of the sample and with a closed-loop piezoelectric stage (ND72Z2LAQ PIFOC objective scanning system, 2 mm travel range, Physik Instrumente, Germany) for the axial displacement of the objective. The fluorescence signal was collected by an independent GaAsP photomultiplier module (H7422, Hamamatsu Photonics, NJ). Emission filters of 482/35 nm and 618/50 nm were used for fibers and cell body detection, respectively.

Cell imaging was performed on a Nikon Eclipse TE300 C2 LSCM (Nikon, Japan) equipped with a Nikon 60x immersion oil objective (Apo Plan, NA 1.4), with Melles Griot (Argon 488 nm) and Coherent (Sapphire 561 nm) lasers. Emission filters for imaging were 514/30 nm and 595/60 nm. Cells were fixed with 4% PFA, rinsed with PBS (+MgCl₂ 0,5 mM, + CaCl₂ 0,8 mM) and permeabilized with 0,075% Triton X. After rinsing with PBS and blocking with 4% BSA PBS, cells were incubated for 20 min with 10 µg/ml biotinylated CTXb, washed and finally labeled with streptavidin Alexa_488 (ThermoFisher, USA, diluted at 1:500) diluted in PBS with 4% BSA. After rinsing again with PBS and water, coverslips were mounted on a glass slide and imaged with LSCM. For LysoTracker™ Red DND-99 (Thermofisher, USA), living cells were grown on 18 mm coverslips and stained following the commercial protocol. Cells were subsequently fixed, permeabilized and labeled with biotinylated CTXb and streptavidin Alexa 488 as described above. For surface labelling, living cells were grown on 18 mm coverslips, rinsed with PBS and incubated with 10 µg/ml biotinylated CTXb diluted in PBS with 4% BSA for 30 minutes on ice, to inhibit endocytic events. After rinsing with PBS, cells were incubated with streptavidin Alexa 488 (1:500) diluted in PBS with 4% BSA for 15 minutes, washed and fixed.