Lsm780 inverted multiphoton microscope

Primary neurons were transfected with pβ-actin-BDNF-mRFP plasmid (kindly provided by Professor Michael A. Silverman, Simon Fraser University, Canada) using Lipofectamine 2000. Movement of BDNF-containing dense core vesicles was evaluated 48 h post-transfection in neurons cultured in phenol red free medium, using a Carl Zeiss LSM780 inverted Multiphoton microscope with an integrated live cell chamber allowing 5% of CO₂ and 37 °C environment, using the 63× objective.
Individual axons were identified according with their distinguishable morphological features, BDNF transport was evaluated in 100 μM length area through time-series of 121 images taken with 1 s interval and exposure of 100 ms from three neurons of different fields, per sample plate (n = 3 plates from 5 different cell cultures, or as indicated in graph bars). Movies were built using ImageJ and analyzed using difference tracker plugin as described by Andrews et al. (2010) (link). Total BDNF moving tracks were accessed as well as the direction of these tracks allowing the evaluation of retrograde or anterograde trafficking.

Generation of ROS in live neurons was evaluated using the probe CM-H₂DCFDA (Invitrogen), according to a previously described method (da-Silva et al., 2004 (link)). Briefly, ROS levels were assessed by incubating neurons 40 min with 2 μM of CM-H₂DCFDA diluted in cell culture media, followed by two rinses with phenol free Neurobasal A medium (Invitrogen) and immediately subjected to live cell imaging analysis. Cells were imaged on Carl Zeiss LSM780 inverted Multiphoton microscope using an integrated live cell chamber allowing the maintenance of a 5% CO₂ and 37 °C environment, images were acquired from 3 fields, randomly chosen, for each experimental condition. Experiment was performed in triplicates and the quantitative analysis performed using Image J (NIH) as described previously (Figueiredo et al., 2013 (link)).
To evaluate the intracellular H₂O₂ content a modification of Zhou et al. (1997) (link) method was employed. Briefly, 30 μg of total protein extract of cell cultures or brain tissue were incubated with 50 μM of the fluorogenic reagent Amplex red (Molecular probes) and 1.0 U/mL of Horseradish peroxidase diluted in 0.1 M of phosphate buffer, pH 7.0, at 37° C. Fluorescence emission was recorded at 587 nm (excitation at 563 nm) for 10 min in Gemini XPS Fluorescence Microplate Reader.

Neurons morphology and neurites length were accessed through immunocytochemistry assays accordingly with the previously described protocol (Ho et al., 2011 ). Briefly, after exposure to rotenone neurons were fixed and permeabilized as described for immunocytochemistry for hyperphosphorylated Tau and MAP-2. Cells were incubated overnight with primary antibody against MAP2 (1/1000) followed by incubation with mouse FITC conjugated secondary antibody for 2 h, mounted with mounting medium containing DAPI and imaged on Carl Zeiss LSM780 inverted Multiphoton microscope using the 20× objective. Images from four randomly selected fields per plate from 3 different cultures were taken and the quantitative analysis of immunofluorescence data was performed using Image J (NIH) and the plug-in NeurphologyJ, as described previously, using MAP2 as a marker for neurites (Ho et al., 2011 ).