We present a protocol for the electron microscopic visualization of the replicative domains in the cell by the EdU labeling of the newly synthesized DNA and label detection with silver enhancement of Nanogold particles and ChromEM staining of the chromatin. This protocol gives the high-contrast pre-embedding labeling with traditional glutaraldehyde fixation that gives the best structural preservation of the chromatin for the room temperature sample processing. This protocol is optimized for the adherence cell growing on the coverslips in the Petri dish.
Add EdU to 10 micro moles final concentration and place the cells into the incubator for the desired labeling time. Fix the labeled cells with 2.5%gluaradehydrate in a 100 millimoles sodium cacodylate solution for one hour. Longer fixation time may adversely affect the EdU labeling.
Remove glutaraldehyde by rinsing the samples with PBS supplemented with five millimoles of magnesium chloride, further regarded as PBS star. Wash three times for 10 minutes. Permeabilize plasma membranes with 1%solution of Trione X-100 in PBS star, make two changes for five minutes each.
This solution is further referred to as PBS star T.Extensively wash the sample with PBS star. Five changes for five minutes each. Quench the residual aldehyde groups with 20 millimoles of glycine in PBS star, two times for 10 minutes.
Place the sample in PBS star with 1%BSA for half an hour. While blocking in BSA, prepare the reaction cocktail for EdU detection. The order of component addition is important.
EdU biotin-azide conjugation is performed in the moist chamber to minimize the evaporation and concentration shifts in the reaction cocktail. Prepare the moist chamber. Place the coverslip on the para-film with a cells facing up and layer 50 to 100 microliters of the reaction cocktail onto the coverslip.
Reaction takes 30 minutes at room temperature. Stop the reaction by washing the sample in PBS star T, five times for five minutes each. Block again with 1%BSA in PBS star T for another half an hour.
Prepare streptavidin-Nanogold solution in PBS star T with 1%BSA. Incubate the sample with streptavidin overnight at plus four degrees in a newly prepared moist chamber. The procedure is the same as with EdU biotin-azide conjugation.
Stop the reaction and wash the sample, five times for 10 minutes each with PBS star T.Stabilize biotin streptavidin complex by additional fixation will 1%glutaraldehyde PBS star for half an hour. Remove glutaraldehyde by intense washing with deionized water. Quench residual aldehyde groups with one milligram per milliliter sodium borohydride.
Wash again thoroughly. Next step is to increase the size of Nanogold particles by a means of silver deposition on the golden course. Prepare premixes to minimize darkroom activity.
Equilibrate the samples with washing buffer and proceed to a darkroom. In the darkroom mix the components to prepare the washing solution and apply it to the samples. Acacia powder solution is highly visicles.
So during mixing the components, rock the tube slowly to avoid bubble and foam formation. The reaction takes from two to five minutes. We recommend to perform a test run to determine the optimal reaction time.
Longer incubation time may result in an unspecific silver deposition. To stop the reaction, quickly rinse the sample with three to five changes of the deionized water. Followed by three more changes for five minutes each.
To protect the silver nanoparticles from dissolution by the osmium tetroxide, incubate the samples in 0.05%tetrachloroauric acid for two minutes in the dark. Wash thoroughly with deionized water. At this stage, additional contrast is added to the DNA containing material.
Saturate the sample with DRAQ5 DNA stain. Add diamionobenzidine solution to the sample and incubate for one minute. Move the coverslip into the glass bottom Petri dish, and place it on the stage of the inverted microscope.
Irradiate several fields of view with 640 nanometer red light. Left panel shows the complete photo bleaching of DRAQ5. On the right panel, the same field of view is imaged in the transmitted light showing DAB precipitate.
Wash samples with deionized water. Next stage should be performed under the fume hood. Prepared partially reduced osmium tetroxide solution.
Be careful. This substance is highly volatile and toxic. Apply the prepared solution onto the sample.
At this stage, the reduced osmium compounds react with diamionobenzidine depositions and increase the contrast of the DNA. Dispose osmium containing solution according to your local regulation and wash samples three times for five minutes each. The sample is further dehydrated in a series of increasing ethanol concentrations followed by the infiltration of the cells with ethanol resin mixes.
Replace alcohol resin mix with freshly prepared pure resin. Fill the properly sized embedding mold with a freshly prepared resin. Place the coverslip with a cells facing down over the resin filled cavity.
Cure the resin for 24 hours at 37 degrees. Then at 60 degrees for at least two days. Remove resin from a bottom surface of a covers.
Drop the slab into a liquid nitrogen and then transfer into boiling water. The glass should detach. Repeat if needed.
Irradiated fields of view should be clearly visible due to the diamionobenzidine deposition and DAB staining. Cut out the area of interest from the slab using the hacksaw or any other suitable instrument. Place the cutout into the ultramicrotome simple holder.
Set up the ultramicrotome for final block trimming. Using the razor blade, prepare the pyramid shaped pillet. The size of a flat area on top of the pyramid should be about 400 by 200 microns.
Mount the prepared pyramid into the ultramicrotome arm and install the knife. Prepare the sections. Section thickness can be adjusted to suit the experimental requirements.
We aim for EM tomography. So we prepared sections with a thickness of 250 nanometers also referred as semi-thin sections. Detach the sections from the knife's edge and mount them on a slot grid.
We used a slot grid, they were round one millimeters to be around, one millimeter side opening. The small opening size provide better stability of the sections. Let the samples dry on the grid.
At this moment, the sections are ready for observation. Place the grid into the electron microscope sample holder. Load the sample into the electron microscope.
Acquire tilt series. Replication foci in mammalian cell nuclei display distinct patterns of distribution depending on the S-phase progression. Successful EdU labeling confirmed by the click reaction with fluorescently labeled azide demonstrates typical replication pattern in the heterogeneous cell population after glutaraldehyde fixation on top.
Streptavidin labeling can be influenced by glutaraldehyde. So first check the fluorescence streptavidin staining under the same condition as streptavidin nanogold on the bottom. Good outcome of the silver enhancements procedure is a dark brown staining of some nuclei and very fainted yellow cytoplast stain, shown on the bottom.
Advantage of a pre-embedding labeling is the possibility to select cells for the sectioning. It is possible at this stage because the labeling patterns become visible under a bright field microscope. The appropriate labeling results in the well demarcated replication sites.
Arrowhead show the DAB stained chromatin fibers. The background labeling on the right is limited to a few nanoparticles per square micron. Semi-thin sections are ready for tomography and do not require addition of gold nanoparticles.
The described approach used for the studies of chromatin organization at sites with enabled replication. For analysis of post replicative rearrangement of chromatin including high resolution imaging of chromatin segregation. It also used for replicated labeling of large chromosomal domains and studies of higher order chromatin folding and heterochromatin.
This imaging technique is also important as a reference point for the data generated by other non microscopic approaches. The method can be also accommodated into correlated microscopic studies including super resolution techniques. This open new horizons in studies of chromatin higher order structure and its dynamics in the various physiological states of the cell.
