Ethanol Fixation and DAPI Staining: A Method to Visualize DNA in C. elegans

研究方案

This protocol is an excerpt from Clarke et al, Manipulation of Ploidy in Caenorhabditis elegans, J. Vis. Exp. (2018).

Tetraploid strains can be validated by counting the number of chromosomes in their unfertilized oocytes. Fluorescent microscopy can be used to screen for the number of chromosome pairs in unfertilized diploid oocytes (prior to the meiotic divisions), if the strain has a fluorescent marker for chromosomes. In the absence of fluorescent chromosome markers, tetraploid nematodes can be screened by fixing the nematodes and staining with a DNA dye; see below for a protocol for ethanol fixing and whole-animal 4′,6-Diamidine-2′-phenylindole dihydrochloride (DAPI) staining.

Whole animal DAPI staining

NOTE: Tetraploid strains carrying 12 connected chromosome pairs can be validated by DAPI staining these animals and counting the number of DAPI bodies in its unfertilized oocytes.

1. Place 5 to 10 µL of M9 buffer on a microscope slide, then transfer 6 - 10 nematodes to the drop.
2. Under a dissecting microscope, draw most of the M9 from the drop without removing the nematodes with a lint-free cleaning tissue. Then, immediately add a 10 µL drop of 90% ethanol onto the worms. Allow the worms to dry completely, but for no longer than a couple of seconds.
3. As soon as the ethanol evaporates (this can be seen as it happens under the dissecting microscope), add an additional 10 µL of 90% ethanol onto the worms.
4. Repeat step 3.1.3 three more times.
5. Once the last drop of ethanol has evaporated, add 6 µL of DAPI or Hoechst dye at the recommended final concentration in the mounting media of choice (for example, a 1:1,000 dilution of a 2 ng/µL DAPI stock concentration in M9). For long-term storage of the slides, 0.5% p-phenylenediamine dissolved in 20 mM Tris-HCl, pH 8.8, in 90% glycerol as an antifade solution, instead of M9 only, may be used.
6. Cover the worms on the slide with a coverslip and seal the edges of the coverslip with nail polish. Score in a fluorescence microscope (step 3.1.7) at least 10 min after adding the coverslip. Slides without antifade can be stored for a few days at 4 °C before scoring, but the quality of fluorescence starts to decline after a few days.
7. Using a fluorescent microscope at 100X magnification, score the number of single DAPI bodies (presumably single chromosome pairs) in the most mature unfertilized oocyte, which is immediately adjacent to the spermatheca and has not yet entered the spermatheca or the uterus.
   1. To score individual DAPI bodies within an oocyte nucleus, use the microscope's fine focus to move slowly from the top of the oocyte nucleus to the bottom during counting. Then, recount the same nucleus while moving the focus in the opposite direction (i.e., from the bottom to the top of the nucleus) to confirm the counted number of DAPI bodies.
8. Score the most mature unfertilized oocyte in each of the two gonads in at least ten hermaphrodites per stable Lon strain. Wild type oocytes have 6 DAPI bodies on average, 5 autosome pairs, and the sex chromosome pair. The presence of 12 DAPI bodies in the oocyte of stable Lon strains indicates that the animals in this strain are either partial (4 sets of Autosomes, 3 X-chromosomes) or complete (4 sets of Autosomes, 4 X-chromosomes) tetraploids.
9. Calculate the average number of DAPI bodies observed from multiple (at least 10) oocytes per strain. Some chromosome pairs will be touching or right on top of one another, so the number of DAPI bodies in an oocyte is often smaller than the actual number of chromosome pairs.
10. (Optional) Further validate stable Lon strains where the average number of DAPI bodies is 12 to test whether they are full (4A, 4X) or partial (4A, 3X) tetraploids by immunofluorescence staining against chromosome axis proteins such as HTP-3. This staining will distinguish chromosome pairs by showing a cruciform pattern from single unconnected chromosomes present in the partial tetraploid.

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材料

Name	Company	Catalog Number	Comments
Dissecting Microscope	Motic Microscopy	SMZ171
Name	Company	Catalog Number	Comments
Staining
Hoechst 33258, Pentahydrate	Biotium	40045
DAPI	Biotium	40011
Name	Company	Catalog Number	Comments
Ethanol Fixation
Ethanol, Pure, 190 Proof (95%), USP	Koptec, VWR	89125-166
Name	Company	Catalog Number	Comments
M9 Buffer
Sodium chloride, Biotechnology Grade	VWR	97061-278
Potassium phosphate, Monobasic Anhydrous Grade	VWR	97062-346
Sodium phosphate, monobasic dihydrate	Fisher Scientific	AC271750025