The overall goal of this procedure is to detect chromatin modifications and DNA sequences simultaneously using 3D confocal microscopy first specific DNA probes are generated. Meanwhile, the experimental cells are fixed perme and immuno stained. For the histone markers of interest.
The cells are then denatured and an overnight fluorescence inside you. Hybridization or fish is performed using one or more DNA probes. Finally, the cells are washed and mounted for 3D fluorescence.
Microscopy analysis of the resulting images reveals nuclear localization of loci of interest and association with specific his stone modifications. The main advantage of this technique is that it combines visualization of nuclear protein, including stones with genomic loci in 3D preserve nuclei, and can help answer key questions in the field of chromatin epigenetic nuclear organization or gene regulation. For this procedure, either backs plasmids or PCR products can be used for labeling.
These should be prepared as described in the accompanying text. Note that for a robust fish signal, the probes should spend at least 10 kilobase pair. Begin this procedure by combining one microgram of DNA and 0.17 micrograms of RNAs A in a micro centrifuge tube.
Incubate for 30 minutes at 37 degrees Celsius. Following the incubation, briefly inactivate the RNAs A on ice and add the appropriate amount of Nick translation. Mix incubate for two hours at 16 degrees Celsius in a refrigerated water bath or a cool block to inactivate the reaction.
Place the tubes at negative 80 degrees Celsius for one hour or negative 20 degrees Celsius overnight. Next, determine the probe size on a 2%agros gel. The smear should be between 101, 000 base pair with the majority at approximately 300 to 500 base pair.
Note that the smear of sci-fi probes is not visible. Once the size has been determined, purify the probes by dialysis on 0.02. Five millimeter filters in two liter beakers filled with water.
Gently pipette 200 micrograms of probe onto the center of each floating filter, then incubate for two hours in the dark. Following the incubation, use a pipette to gently transfer the probe from the filters to a fresh micro centrifuge tube. Next, add blocking factors to the probes as follows.
One microgram of CO 1D NA.One microgram of high block DNA and 10 micrograms of salmon sperm for one microgram of DNA probe and mix store the DNA probes at negative 20 degrees Celsius. On the day of the fish procedure, the probes are precipitated and prepared for use. Note that the steps described in this section of the video should be performed in parallel with the steps in the following section.
To precipitate the probes, follow them and combine them with 0.1 volume of three molar sodium acetate at pH 5.2 and 2.5 volumes of 100%Ethanol incubate for one hour at negative 80 degrees celsius. After the incubation, spin the probes down at 16, 000 times G at four degrees Celsius for 30 minutes. Following the spin, discard the supernatant and air dry the pellets in the dark.
Depending on the amount and color of the probes, the pellet may or may not be visible. Resus suspend the pellet in 15 microliters of hybridization buffer per test condition and shake using an einor thermo mixer in the dark at 37 to 45 degrees Celsius for approximately 20 minutes. Next to denature the probes incubate them for five minutes at 75 to 95 degrees Celsius.
Then to pre probes, incubate them at 37 degrees Celsius for 30 to 60 minutes on the thermo mixer. Immediately following the incubation, the probes will be applied to cells on cover slips as described in the next section of the video. Note that for the following steps, incubation times should be modified as necessary for the antibodies and cells used.
The steps described in this section of the video should be performed in parallel with the steps in the previous section as described in the accompanying text. Drop non-adherent cells on top poly lysine coated cover slips in wells of six well plates and let them attach for five minutes. Then gently add two milliliters of 2%paraform aldehyde in PBS incubate for 10 minutes at room temperature following the incubation.
Rinse each well with PB S3 times then to permeable the cells, add two milliliters of ice cold, 0.4%Triton X 100 in PBS and incubate for five minutes on ice. After aspirating the permeable solution if DNA fish will be combined with immunofluorescence, proceed as follows. First pipette 100 microliters of blocking solution onto param in a humidified chamber.
Place the cover slip cell side down onto the blocking solution and incubate for 30 minutes at room temperature. Next, transfer. The cover slips to 100 microliter drops of the primary antibody diluted in blocking solution.
Here the antibody used recognizes phosphorylated Syrian 1 39 of H two A x or gamma H two A x. Incubate the slides in a humidified chamber for one hour at room temperature. After the incubation, use forceps to carefully remove the cover slips.
If the cover slips are difficult to pick up, flood with PBS and try again. Transfer each slide to a well of a six. Well plate containing two milliliters of 0.2%BSA 0.1%tween 20 in PBS wash the cover slips in this solution three times for five minutes at room temperature with shaking, then incubate cells with the appropriate fluorophore conjugated secondary antibody diluted in blocking solution for one hour at room temperature in a dark and humid chamber on param.
As before following the incubation, rinse the cells in 0.1%tween 20 in PB S3 times for five minutes at room temperature in the dark to postfix the cells, submerge them in two milliliters of 2%para formaldehyde in PBS for 10 minutes at room temperature in the dark. Once the cells have been stained or if they are not stained, continue DNA fish by rinsing the cover slips three times in PBS. Then place the cover slips cell side down onto a 100 microliter drop of 0.1 milligrams per milliliter.
RNAs on param in a humidified chamber and incubate for one hour at 37 degrees Celsius. After the incubation, rinse the cover slips three times in PBS then perme the cells in ice cold 0.7%Trix 100 in 0.1 molar hydrochloric acid for 10 minutes on ice in the dark. After three rinses in PBS denature the cells in 1.9 molar hydrochloric acid for 30 minutes at room temperature in the dark and wash three more times in ice cold PBS next to hybridize cells with the probes.
Place the cover slips cell side down onto a 15 microliter drop of probe prepared in the previous section of this video on a microscope slide, seal the slide with rubber cement and incubate overnight at 37 degrees Celsius in a dark humidified chamber. The next day, use forceps to carefully remove the rubber cement from the slides. Then flu the cover slips with two Acts SSC and use forceps to carefully remove the cover slips with the cells.
Then place them in wells containing washing solution. Next, perform the following washes with shaking in the dark two XSSC for 30 minutes at 37 degrees Celsius, two XSSC for 30 minutes at room temperature and one XSSC for 30 minutes at room temperature to mount the slides, place the cover slips cell side down onto a 10 to 15 microliter drop of prolonged gold mounting medium containing 1.5 milligrams per milliliter DPI seal with nail polish. The slides can be stored for months at negative 20 degrees Celsius.
Once the slides are ready, collect 3D images with optical sections separated by 0.3 microns and analyze as described in the accompanying document to determine whether changes in association of the two alleles of the antigen receptor loci are linked with the regulation of the process of VDJ recombination during lymphocyte development, wild type and mutant cells deficient in the recombinase protein rag one were used to examine a locus of interest using a DNA fish probe generated from a back that spans approximately 200 kilobase pair encompassing the genomic region. Distances between alleles were measured between the center of mass of each back signal in 3D image stacks dashed circles, outline nucleus shapes. The scale bars shown are one micron cumulative frequency distribution curves were then generated to show the entire range of distances between the two alleles measured in the wild type and mutant cells.
The non-parametric two sample KS test was then used to assess the significance of the differences between the two distributions. The range of the most robust differences in inter allelic distances was assessed using a series of two tail fisher exact tests indicated by the blue dots. The most robust P values indicated by the higher dots were found for the distances around one micron.
Thus, the cutoff for close association or pairing of the two alleles was set up at one micron. Cumulative frequency curves shown here indicate the range of distances for close association of the two alleles from zero to 1.5 microns in the two different biological conditions. The significance of pairing of the two alleles was assessed using the two tail fisher exact test for a cutoff at one micron.
This graph shows that in the mutant condition, the two alleles are not associated with high frequency as in the wild type. Thus, the missing protein rag one is involved inic pairing. After watching this video, you should have a good understanding of how to combine immunofluorescence and DNA fish.
This technique paved the way for researchers by giving insights into the global changes in nuclear architecture, the behavior of specific genomic loci and regions within the nuclear space and the association of specific nuclear proteins within these regions. And it helps understanding the mechanisms underlying different disease following this procedure. Other methods like genome wide ice throughput techniques such as chromatin, immuno precipitations, or chips seek or chromosome capture confirmation coupled with deep sequencing like fore five C or IC, can be performed to provide global data on cell populations.
