Fluorescence in situ hybridization protocol in human sperm nuclei has been widely incorporated in the field of clinical diagnosis. One of the applications of this methodology is spermatozoa ploidy screening. As part of the advice on reproduction given to infertile males, the presence of numerical abnormalities is higher in these individuals than in the general population, and they often display abnormal seminal parameters.
This represents an increased risk of transmitting these anomalies to any offspring. Today we are going to illustrate the fluorescence in situ hybridization protocol in decon condensed sperm nuclei for the analysis of chromosomes 13 18 21 x and Y.Once the sample has been collected, it should be left at room temperature for 20 minutes until liqui action occurs. To begin the protocol, the sample must be transferred to a centrifuge tube.
The solutions we are going to use in the first part of the experiment are hypertonic preheated at 37 degrees C and methanol acetic acid in the proportion of three to one. These must be freshly prepared. Then the sample is ready to centrifuge at a thousand Gs for five minutes.
Now, gently remove the supernatant and add the hypertonic solution Drop by drop until you have a volume of about 10 milliliters. This tube must remain at 37 degrees C for 30 minutes, and then the sample must be centrifuged again. Centrifugation causes the spermatozoa to collect at the bottom of the tube, allowing us to replace the hypotonic solution with the methanol.
Acetic acid centrifugation must be repeated to change the methanol acetic acid as many times as is necessary to obtain a white pellet. When this occurs, more methanol acetic acid must be added. The final volume should be adjusted according to the cellular dispersion that you want to obtain in further extensions.
The final step is to make the extensions by dropping the fixed sample onto Reed slides stored in methanol at minus 20 degrees C.It is helpful to deli the area of the slide containing the sperm extension for prospect re localizations. This can easily be done using a diamond pencil on the other side of the slide. Once an optimal cellular dispersion has been verified, the slides should be stored at minus 20 degrees C for a minimum of 24 hours.
Before proceeding with the protocol. In this part of the experiment, we are going to use saline sodium citrate solution ethanol series DRE fatal and fide. When the slides have been defrosted, the fixed samples must be washed and dehydrated by submerging them in two consecutive coplan jars containing two times SSC.
This will take three minutes for each coplan. Then the slides must be submerged for two minutes in each of the ethanol coplan jars in order of increased concentration. At this point, in order to continue with the following stages, the slides must be dried out.
Given that the DNA in the sperm nucleus is highly compacted, it is necessary to decon condense the chromatin before denaturing it. This can be done using a DTT solution at 37 degrees C.This is a product that acts by breaking the sulur bridges of the protamine that coil the DNA in the spermatozoa nucleus. The incubation time of the slide in DTT must be adjusted according to the reactivity of the semen sample to this product.
Usually this is around eight minutes, although it can vary from two to 15 minutes after this time, the slides must be transferred immediately to two times SSC for three minutes and again for a further three minutes, followed by two minutes in each of the ethanol coplan jars. For the probe hybridization, it is essential to work with single DNA strands. This requires a denaturation stage before the addition of the probes involving an incubation period of five minutes in a former mite solution at 73 degrees C.To conclude the slide treatment, a final transfer in the ethanol solution is required submerging the slides for one minute per coplan jar.
The slides are then ready to be hybridized in the fish studies carried out for clinical diagnosis. The most commonly analyzed chromosomes include the sex chromosomes and the chromosomes 13, 18, and 21. To analyze these chromosomes, we will use a multicolor DNA probe kit from vices.
This contains one vial with a probe combination for the alpha satellite region of chromosomes, X, Y, and 18 labeled green origin and aqua respectively, and another vial with a combination of probes for chromosomes 13 and 21 labeled green and orange respectively. The volume of the probe mixture added will vary according to the size of the area you want to hybridize as a reference for a 15 by 15 cover slide. A volume of five microliters is sufficient.
Both probe mixtures are pre denatured in a hybridization buffer for easy use, so you can add them directly to the deacon condensed and denatured slides. There are also other commercial probes available for a wide range of chromosomes and Loki that can be used in these experiments. Usually, however, these probes require a pre denaturing treatment before the addition of the mixture to the target sample.
In these situations, it is sufficient to follow the manufacturer's instructions. The targeted region must be covered with a cover slide and sealed with rubber cement. Then the slides are ready to be placed into the hybridization chamber, pre warmed to 37 degrees C and incubated at that temperature for six to 24 hours.
In the final part of the protocol, we are going to use these two wash solutions made up of different concentrations of saline sodium citrate solution and the detergent NP 40. After the incubation time, the slides can be removed from the hybridization chamber to eliminate the rubber cement. The then carefully take off the cover slide.
As shown, the slides are submerged for two minutes In the first wash solution, pre-warned to 73 degrees C.Then they're transferred to the second wash solution at room temperature for a further minute. These two washes will help us eliminate any unspecific hybridization signals. After these two washes, we have to wait for the slides to dry out to conclude the last step of the protocol.
At this point, a counter staining product is added to facilitate the visualization of the nucleus of the cells. One of the most common products used for this purpose is dpi. The volume added must also be adjusted to the size of the hybridized area as a reference for an 18 by 18 cover slide.
A volume of eight microliters of DPI is sufficient. The cover slide can be sealed with nail varnish, and now the preparations are ready to be visualized. Otherwise, they can be stored at minus 20 degrees C until their prospect analysis.
The microscope that we are going to use for the visualization is an Olympus BX 60 epi fluorescent equipped with a triple band filter for dpi, Texas Red, FITC, and single band filters for aqua FITC and Texas Red through the triple band filter, the preparation hybridized with x, Y, and 18 display signals for these three chromosomes. Using the single band filter for aqua, we can only visualize fluorescent signals for chromosome 18. We should expect one of these signals in every normal spermatozoa.
However, when using the single band filter for FITC only, some spermatozoa show a fluorescent green signal corresponding to the X chromosome. The spermatozoa that lack the green signal should display a red signal for the Y chromosome, which could be specifically visualized through the filter for Texas red Visualizing the 1321 hybridization through the triple band filter. We can distinguish signals for these two chromosomes.
The green signal corresponds to chromosome 13 and can be specifically visualized using the single band filter for FITC. The single band filter for Texas Red allows us to specifically visualize the signals for chromosome 21. All normal spermatozoa should display one signal for each one of these two probes.
So to sum up, here are the main stages of the protocol. Again, the fixation process should be accurately carried out to obtain good quality extensions with a drop by drop addition of the methodol acetic acid. To avoid the formation of sperm aggregates in the de condensation process, the incubation time in DTT solution must be adjusted according to the reactivity of the semen sample to this product.
Excessive exposure of the spermatozoa to DTT would result in disperse signals at the end of the protocol, whereas insufficient exposure would result in zero probe hybridization and a lack of some signals. Whereas the denaturation of the sperm sample is mandatory, the probe denaturation must be adapted to the manufacturer's indications. It is important to maintain strict control over the time and temperature of the two post hybridization washes.
A higher temperature or an excessive wash time can result in the elimination of some signals, whereas the opposite would not eliminate unspecific signals. Finally, the use of an epi fluorescent microscope equipped with adequate specific filters is essential for good visualization and signal interpretation. And that's it.
Thank you for watching and good luck with your experiments.
