Time-Lapse Imaging to Monitor the Transcellular Spreading of Prion-Like Proteins in Transgenic Nematodes

Protokol

1. Monitoring Transcellular Spreading of Prion-like Proteins By In Vivo Time-lapse Imaging

NOTE: Grow C. elegans wild-type (WT) (N2) and transgenic lines according to standard methods and carefully control the cultivation temperature.

1. Generate transgenic lines of C. elegans expressing the prion-like protein, tagged with a monomeric red fluorescent protein (mRFP).
2. Prepare synchronized populations by egg laying or bleaching according to standard methods.
   1. Synchronization by egg-laying
      1. Transfer 10 - 20 gravid adults on a plate and let them lay eggs for 1 - 2 hr. Remove adults from the plate and let the progeny grow until the desired age.
   2. Synchronization by bleaching
      1. Collect an unsynchronized population of gravid adults and bleach them with an alkaline hypochlorite solution (250 mM sodium hydroxide (NaOH) and 1:4 (v/v) dilution of commercial bleach in water (H₂O)). Wash the eggs twice (218 x g for 1 min) with M9 buffer (21 mM Na₂HPO₄·7H₂O (di-sodium hydrogen phosphate heptahydrate), 22 mM (potassium dihydrogen phosphate) KH₂PO₄, 86 mM sodium chloride (NaCl), 1 mM MgSO₄·7H₂O (magnesium sulfate heptahydrate), add dH₂O up to 1 L).
      2. Allow them to hatch in M9 buffer with gentle agitation overnight (O/N) at 20 °C. Worm development will be arrested at the L1 stage in the absence of a food source, leaving a synchronized population. Transfer L1s onto fresh Nematode Growth Media (NGM) plates seeded with OP50 E. coli bacteria and let the progeny develop until the desired age.
3. Prepare 2% agarose pads (in H₂O) on a microscope slide as described.
   1. Prepare two microscope slides with labeling tape placed over their entire length to be used as spacers. Place a third microscope slide between them.
   2. Dissolve 2% agarose in H₂O and pipette one drop onto the clean slide.
   3. Place a fourth slide perpendicular to the three other slides on top of the agar drop. Gently press it down to flatten the pad to the same thickness as the labeling tape.
   4. Let it dry for 1 minute before removing the spacers and gently pulling the slides apart. The agar pad will stick to one of them.
4. Pipette ~10 µl anesthetic (2 mM levamisole in M9 buffer) to the pad and transfer ~10 animals using a platinum wire pick. Cover with a cover slip (~22 x 22 mm) and take images within 1 hr.
5. Alternatively, to acquire movies over a longer period of time or to further reduce the possibility of any movement of the animals, use a combination of anesthetic and bead immobilization.
   1. Prepare 10% agarose pads (in M9 buffer) as described and add worms to 3 µl nanosphere size standards solution (polystyrene beads, 100 nm) plus 3 µl anesthetic (4 mM levamisole in M9 buffer). Cover gently with a cover slip. To avoid desiccation, seal the coverslip with VALAP (a mixture of equal amounts of Vaseline, lanolin, and paraffin wax).
6. Image immobilized worms using a confocal microscope.
   ​NOTE: Results are obtained using a Spinning Disc AF Confocal Microscope equipped with an EM-CCD camera and Microscopy Automation & Image Analysis Software such as MetaMorph outline the specifics below, but other comparable confocal imaging systems can also be used.
   1. Use the 63X or 100X/1.4NA oil objective and place the microscope slide containing worms into the microscope slide holder.
   2. Open the software. Adjust laser power and filters for mRFP imaging. Use the 561 nm laser at 10% power and emission filter >600 nm.
   3. Open "MultiDimensional Acquisition". Under the "Main" tab, check the boxes for "Timelapse" and "Run Journals" (Hardware Auto Focus: off).
   4. Under the "Saving" tab, select or create the directory folder where the files should be saved. Assign a name to the file.
   5. Under the "Wave Length" tab, select the appropriate illumination and adjust exposure and gain. Use "YokoQuad Red" (or an equivalent illumination setting for mRFP imaging) with an exposure between 100 and 300 msec and a camera gain between 100 and 300, depending on the individual sample (assessed by using the live image).
   6. Under the "Timelapse" tab, select "Number of time points" = 301, "Duration" = 5 min, and "Time/Interval" = 1 sec.
   7. Under the "Journals" tab, select Journal: "AFC SET Z HOLD" and "AFC Return to Z HOLD", Type: "Special" (twice), Initial Point: "Start of time point" and "End of time point". This autofocus option is important to image the same section over a longer period of time.
   8. When the setup is complete, press "Acquire." The timelapse video is saved as separate TIFF files.

Malzemeler

Name	Company	Catalog Number	Comments
Reagent
Nanosphere size standards 100 nm	ThermoScientific	3100A
Levamisole	Sigma	L-9756
Equipment
Sorvall Legend XTR Refrigerated Centrifuge, 120VAC	ThermoScientific	75004521	http://www.coleparmer.com/Product/Thermo_Scientific_Sorvall_Legend_ XTR_Refrigerated_Centrifuge_120 VAC/EW-17707-60
96 pin replicator	Scionomix		http://www.scinomix.com/all-products/96-pin-replicator/
HiGro high-capacity, incubating shaker	Digilab		http://www.digilabglobal.com/higro
Multidrop Combi Reagent Dispenser	Titertrek		http://groups.molbiosci.northwestern.edu/hta/titertek.htm
Biomek FX AP96 Automated Workstation	Beckman Coulter		http://groups.molbiosci.northwestern.edu/hta/biomek_multi.htm
Innova44 shaker	New Brunswick		http://www.eppendorf.com/int///index.php?sitemap=2.3&pb=d78efbc05310ec 04&action=products&contentid=1& catalognode=83389
M205 FA	Leica		http://www.leica-microsystems.com/de/produkte/stereomikroskope-makroskope/fluoreszenz/details/product/leica-m205-fa/
ORCA-R2 C10600-10BDigital CCD camera	Hamamatsu		http://www.hamamatsu.com/jp/en/community/life_science_camera/product/search/C10600-10B/index.html
Spinning Disc AF Confocal Microscope	Leica		http://www.leica-microsystems.com/products/light-microscopes/life-science-research/fluorescence-microscopes/details/product/leica-sd-af/
Falcon 4M60 camera	Teledyne Dalsa		http://www.teledynedalsa.com/imaging/products/cameras/area-scan/falcon/PT-41-04M60/
Software
MetaMorph Microscopy Automation & Image Analysis Software	Molecular Devices		http://www.moleculardevices.com/products/software/meta-imaging-series/metamorph.html
Hamamatsu SimplePCI Image Analysis Software	Meyer Instruments		http://meyerinst.com/imaging-software/hamamatsu/index.htm
ImageJ	NIH		http://rsbweb.nih.gov/ij/download.html
wrMTrck plugin for ImageJ			http://www.phage.dk/plugins/wrmtrck.html
C. elegans strains
N2 (WT)	Caenorhabditis Genetics Center (CGC)		http://www.cgc.cbs.umn.edu/strain.php?id=10570