The aim of this protocol is to visualize heterochromatin aggregates in Drosophila cells. To achieve this, we use polytene cells which are found in the salivary glands of third instar larvae The genome of these cells is amplified nearly a thousand times, and the chromosomes conserve the features of interface cells. They have well-defined telomeres, and bridges called chromocenter in which all the centromeres aggregates, and it's mainly heterochromatic.
Using the third instar larvae tissue, also allow us to evaluate change in the heterochromatic aggregates in different genetic mutants'background. To optimize the third instar larvae culture, you will need first to collect 5 to 10 days old adults, and put around 50 in a broad neck bottle of a standard media. 25 male and 25 female.
Place the bottle in a controlled temperature incubator at 25 Celsius degrees for 12 hours. After the incubation time is over, remove the adults and transfer them to a new bottle to repeat the procedure. Let the embryos grow at 18 degrees Celsius for 22 hours.
For larvae collection, make sure you choose the wandering larvae, which do not have overt sphericals. Dissect 15 pairs of salivary glands, or as much as you can in 30 minutes period in cold PBS with protest inhibitors under the microscope. Transfer the salivary glands to another tube with ice cold PBS.
Wash once with cold PBS plus protests inhibitors. Remember, always wait for the tissue to reach the bottom of the tube. After removing the PBS from the last step, add directly 0.5 milliliters of one X group gum fixation buffer and 50%methanol plus 2%formaldehyde.
Incubate for two hours at four Celsius degree with mild rotation. Carry out one five minute rotation wash with Tris-Triton buffer, adding one milliliter. Always wait for the tissue to reach the bottom of the tube.
Note:wait for the tissue to reach the bottom of the tube. Incubate the salivary glands with Tris-Triton, the same as above, plus 1%of beta mercaptoethanol for two hours at 37 degrees Celsius with mild shaking. Wash with BO3 buffer, and incubate with BO3 plus 10 millimolar DTT at 37 degrees Celsius with mild shaking for 15 minutes.
At the end of the incubation period, perform a wash with one milliliter of BO3 buffer alone. Incubate the salivary glands in one milliliter of buffer B for two hours at room temperature with rotation. Remove all buffer B and add buffer A plus antibody of interest.
In this case, we use HP one, A C 1 8 9 from Iridium bank. 1 in 3, 000 over night at four degrees with rotation. At this point is important that the shaking does not raise bubbles, which might damage the antibody.
Give treat by 15 minutes washes with buffer B, under stirring at room temperature using one millimeter each time. The glands are transferred to buffer B together with the secondary antibiotic couple to a floor refer for two hours, under rotation at four degrees. At this step, it's important to cover the tube with aluminum paper foil to protect the secondary antibody from the light.
Carry out 2x15 minute washers at room temperature, while rotation with one milliliter of buffer B.Incubate with a DNA marker such as Sydox or OH, and dissolve in one milliliter of buffer B for 10 minutes at room temperature with rotation. Carry out one wash with buffer B and once with PBS, each wash lasting 10 minutes, while rotating at room temperature. Finally, mount the salivary glands on a slide, making a pool with a cover slip.
Put the salivary glands in the middle of the pool and cover with Sidiflor. To avoid deformation of bubble, extending the viscous liquid all over the place. Then, seal all the slides with clear nail polish.
Observe under a fluorescence or confocal microscope. If the sample is not going to be observed on the same day, then store from the light at four degrees. Representative results of HB1 monostaining in Drosophila salivary glands are shown in figure one.
A positive result is to observe one focal point like in A, athero chromatic aggregate or condensate. A negative result is no signal or a dispersed signal. Sometimes, a double signal can be observed like a double point in C, but usually occurs in smaller quantities.
To analyze the data they can be represent as a bar graph, comparing the distribution of HP1 with different mutant backgrounds. For example, in figure two, we can see that 98%of nuclei present a distribution of one point. And 2%of two foci in wild type.
In the mutant, the proportion changed, and the presence of two foci increased to 40%Figure three shows representative trimethylation lysine 9H3 in monostaining. Result in Drosophila salivary glands observing one focal point in B, it's a favorable result at the chromatic aggregate or condensate, a double or triple signal in C can seen on rare occasion, but in a small quantities. At the end of this protocol, you will be able to see a diachromatic condensates of HP1 protein despite state limitations, it will be a good first approach.
