Dfc345 fx camera

Live-cell visualization of whole neurons was performed by transfection using pLV-hSyn-RFP plasmid from Edward Callaway (Addgene plasmid # 22909) and Lipofectamine 2000 (Thermo Fisher Scientific) in accordance with the manufacturer’s protocol. Transfections were carried out in serum-reduced OptiMEM media (Thermo Fisher Scientific). A Leica DM6000 FS microscope, Leica DFC 345 FX camera, and the Leica Application Suite software were used to produce micrographs. We performed live-cell visualization of 2 neurons from two different preparations in order to illustrate our experimental approach (Figures 3a, 4a,e and 5a and Figure 2—figure supplement 1a,b) as well as to demonstrate that the spatiotemporal distribution of the recorded signals matches with neuronal morphology (Figures 1a, 2, 3a, 4e and 5a and Figure 2—figure supplement 1a,c). The latter has also been demonstrated in previous studies, where live-cell visualization was used to verify axonal AP propagation tracking by means of HD-MEA-based monitoring of extracellular APs (Bakkum et al., 2013 (link); Radivojevic et al., 2016 (link)).

Z-stack pictures of protoperithecia (female structures) were taken using a Leica M205 FA stereomicroscope with a Leica DFC345 FX camera. Representative images of asci (spore sacs) and perithecia (fruiting bodies) were obtained using an Apple iPhone 5 with a Magnifi photoadapter (Arcturus Labs, Palo Alto, CA) on a Vanguard 1231CM microscope. An Olympus BX61 was used for fluorescent microscopy. Preparation and visualization of asci (≥10/cross) were performed as described (Alexander et al. 2008 (link); Xiao et al. 2010 (link)), with similar exposure times used across samples (50–150 ms for DAPI and 500–800 ms for fluorescent proteins).

For neuron survival assays, we quantified the number of STX1-null hippocampal neurons transduced with Stx1a, Stx2, and GFP (as control) at DIV 15, 22, and 29, and compared this to the number of neurons at DIV8. Also, 2 wells per group were plated and 15 random ROIs of 1.23 mm² per well were imaged at the different time points (30 total ROIs per group at each time point per culture). Phase-contrast bright-field images and fluorescent images with excitatory lengths of 488 and 555 nm were acquired on a DMI 400 Leica microscope, DFC 345 FX camera (Leica), HCX PL FlUPTAR ×10 objectives (Leica) and LASAD software (Leica). The neurons were counted offline with the 3D Object Counter function in Fiji software (Vardar et al., 2016 (link)). For the example images, the cultures were fixed at the corresponding time points in 4% paraformaldehyde (PFA) in 0.1 M phosphate-buffered saline (PBS), pH 7.4 , and immunocytochemistry was done as follows.

Chromosome preparations were obtained from male gonads. Testes were dissected in a drop of 45% acetic acid and squashed. The coverslips were removed using dry ice. Prior to FISH treatment, the preparations were examined using phase contrast microscopy.
FISH with (TTAGG)_n and 18S rDNA probes was applied as previously reported for some other insects (Kuznetsova et al. 2015 , Maryańska-Nadachowska et al. 2016 (link), Golub et al. 2016 ). In brief, chromosome preparations were treated with 100 µg/ml RNase A and 5 mg/ml Pepsin solution to remove excess RNA and proteins. Chromosomes were denatured on a slide in a hybridization mixture with biotinylated 18S rDNA probe from the genomic DNA of Pyrrhocorisapterus (Linnaeus, 1758) and rhodaminated (TTAGG)_n probe with addition of salmon sperm DNA and then hybridized for 36 h. Hybridization signals were detected with NeutrAvidin-FITC.
Chromosomes were mounted in antifade medium (ProLong Gold antifade reagent with DAPI; Invitrogen) and covered with a glass coverslip. Chromosome slides were analyzed under a Leica DM 6000 B microscope. Images were taken with a Leica DFC 345 FX camera using Leica Application Suite 3.7 software with an Image Overlay module.