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[…] Standard documentation was performed with AxioCam monochrome digital cameras (Carl Zeiss Ltd.) mounted on BX50WI or BX51 Olympus compound fluorescent microscopes. Z-stacks of embryonic CNSs were taken with a Leica DM6000 B microscope and extracted with Leica MM AF Premier software. Z-stacks of adult fly brains were taken with a Leica DM6000 B microscope or with a 3i Marianas Spinning Disk Confocal Microscope. Using custom software written in Python and NumPy, fly brain images taken with a Leica DM6000 B microscope were individually band-pass filtered (A trous wavelet [1][2], linear 3x3 filter, keeping scales 1–4) to remove stationary background.To quantify the number of synaptic densities in mature neurons in culture and the number of vesicles containing synaptic proteins in 8h neurons in culture, we used ImageJ (RRID:SCR_003070). In detail, we used thresholding to select synaptic densities from axons of single isolated cells, followed by particle analysis. For all experiments done in parallel, identical thresholds were used. For the quantification of synapses in mature neurons in culture, we selected polarised neurons with a clear distinguishable axon, the same neurons were used to study axon length and number of branches.To quantify synaptic proteins or Unc-104 in the soma of neurons, we manually selected the area of the somata using the tubulin or HRP channel and measured the signal intensity derived from the Syt or Unc-104 channel. To measure the levels of Unc-104 at the tip of axons, we selected an area of the same size at the most distal part of axons and measured the signal intensity derived from the Unc-104 channel. To quantify synaptic proteins at the tip of embryonic motorneurons in vivo. we manually selected the area occupied by the growth cones using the FasII staining and measured the signal intensity derived from the Syt channel; the background intensity was subtracted. Images used for these measurements did not contain saturated levels. Also, to measure the number of synaptic densities in DC neurons in the medulla of the adult brain, we used thresholding to select synaptic densities followed by particle analysis. The number of branches in the medulla per DC neuron was quantified manually. To quantify MT stability upon nocodazole treatment, we counted the number of breaks in the microtubule bundle per axon.Time lapse imaging of cultured primary neurons (in Schneider's/FCS) was performed on a Delta Vision RT (Applied Precision) restoration microscope using a 100x/1.3 Ph3 Uplan Fl objective and the Sedat filter set (Chroma 89000). The images were collected using a Coolsnap HQ (Photometrics) camera. The temperature control was set to 26°C. For time lapse recording, images were taken every 2 s for 2 min. To generate transport measurements, vesicles containing fluorescently tagged Syt were tracked manually using the manual tracking plugin for ImageJ. […]

Pipeline specifications

Software tools Numpy, ImageJ
Applications Miscellaneous, Laser scanning microscopy, Microscopic phenotype analysis
Organisms Drosophila melanogaster