This method is suitable for live imaging of cytoplasmic dynamics using frog egg extracts, enabling the study of spatial pattern information in the cytoplasm. Samples can be shaped into a layer with a uniform and adjustable thickness and imaged across a 2D field or 3D volume. This method is cost effective and easy to implement.
Begin by applying a layer of FEP adhesive tape to a glass slide with a roller applicator. Cut off excessive tape over the edges with a clean razor blade. Next, prepare FEP tape coated cover slips in the same way.
Then apply a double-sided sticky imaging spacer to the FEP tape coated side of the slide leaving the protective liner unpeeled. Transfer eggs to a 400 milliliter glass beaker and remove as much egg-laying buffer as possible by decanting. Then incubate the eggs in 100 milliliters of freshly prepared dejellying solution and gently swirl them.
After about three minutes, pour off the solution and add 100 milliliters of fresh dejellying solution. Continue the incubation until the eggs are tightly packed, but avoid leaving eggs in the dejellying solution for more than a total of five minutes. After the incubation, remove the dejellying solution as much as possible and wash the eggs in 0.25x MMR buffer.
Swirl the eggs, then pour off the buffer. Repeat a few times until a total of one liter of the buffer is used for the washes. Next, wash the eggs a few times with a total of 400 milliliters of egg lysis buffer, removing abnormal eggs between the washes.
Then use a transfer pipette with a wide cut tip to transfer the eggs to a 17 milliliter round bottom centrifuge tube containing one milliliter of egg lysis buffer. Spin the tube in a clinical centrifuge at 400 g for 15 seconds to pack the eggs. Then remove as much of the egg lysis buffer as possible using a Pasteur pipette.
Determine the approximate volume of the packed eggs. then add five micrograms per milliliter each of aprotinin, leupeptin and cytochalasin B and 50 micrograms per milliliter of cyclohexamide directly on top of the packed eggs. Crush the eggs by centrifusing the tube for 15 minutes at 12, 000 g and four degrees Celsius in a swinging bucket rotor.
Next, attach an 18 gauge needle to a syringe. With the needle tip bevel facing up puncture the tube from the side at the bottom of the cytoplasmic layer and recover the extract by drawing slowly. Transfer the recovered cytoplasmic extract to a new micro centrifuge tube and keep it on ice.
When ready to image, supplement the extract with the desired reagents and fluorescence imaging probes. Remove the top protective liner from the imaging spacer on the prepared slide and deposit approximately seven microliters of extract at the center of the well. Immediately apply the FEP tape coated cover slip with the FEP side facing the extract to seal the well and quickly proceed to imaging.
Set the slide on an inverted or upright microscope with a motorized stage and a digital camera. Image the extracts at the desired spatial positions in time intervals in both bright field and fluorescence channels. A time-lapse experiment using xenopus laevis egg extracts to study the self-organization of the cytoplasm during interphase is shown here.
After about 20 minutes of incubation at room temperature areas depleted of light scattering cytoplasmic components were visible in both bright field and mitochondria channels. By about 60 minutes at room temperature, a spatial pattern consisting of cell-like compartments should be well established with microtubules forming a hollow wreath-like structure and mitochondria clearly partitioned into each compartment. A comparison of extract performance in imaging chambers with and without FEP tapes on glass is shown here.
In the chamber made with FEP taped glass the extract self-organized into normal cell-like patterns. In the chamber where the glass surfaces were not covered by the FEP tape, the extract showed abnormal bright field and microtubule patterns that became increasingly disrupted over time. No significant differences were observed in the nuclear import of the GST-mCherry-NLS protein.
The important things to remember are to passivate the glass surfaces with FEP tape and to remove all bad eggs during extract preparation. With this system, we can easily control the thickness of the extract sample, paving the way for imaging cytoplasmic patterns in 3D, using light sheet microscopy.
