The authors acknowledge Marjo van Brakel, Dominique Smeets, Guillaume van de Zande, Patricia van Cleef and Milan Intezar for their expertise and technical assistance. Funding sources: Merck Serono (A16-1395), Goodlife, and Ferring.

The TurnerFertility study protocol has been approved by the Central Committee on Research Involving Human Subjects (NL57738.000.16). In this study, the ovarian cortex tissue of 93 females with TS was obtained. Materials that require safety precautions are listed in Table 1.
Table 1: Safety precautions.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Material</td>
			<td colspan="3">Hazard</td>
		</tr>
		<tr>
			<td>Acetic acid</td>
			<td colspan="3">Severe skin burns and irritation of the respiratory system</td>
		</tr>
		<tr>
			<td>Collagenase</td>
			<td colspan="3">Irritating to the eyes, respiratory system and skin</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td colspan="3">Irritating to the eyes, respiratory system and skin</td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td colspan="3">Irritating to the eyes, respiratory system and skin</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td colspan="3">Highly flammable</td>
		</tr>
		<tr>
			<td>Formaldehyde</td>
			<td colspan="3">Toxic after inhalation, ingestion and skin contact</td>
		</tr>
		<tr>
			<td>Formamide 
			(in fluorescence probes)</td>
			<td colspan="3">May harm the unborn child</td>
		</tr>
		<tr>
			<td>Liberase</td>
			<td colspan="3">Irritating to the eyes, respiratory system and skin</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td colspan="3">Highly flammable, toxic by inhalation, ingestion and skin contact</td>
		</tr>
		<tr>
			<td>Nonidet P40</td>
			<td colspan="3">Irritating to the skin or eyes</td>
		</tr>
		<tr>
			<td>Pepsin</td>
			<td colspan="3">Irritating to the eyes, respiratory system and skin</td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td colspan="3">Breathing difficulties after inhalation</td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td colspan="3">Highly flammable, toxic after inhalation and skin contact. Avoid contact with the eyes.</td>
		</tr>
	</tbody>
</table>
Table 1: Materials that require safety precautions.
1. FISH on isolated individual ovarian cortex cells
<ol>
	<li>Dissociation of ovarian cortex tissue to obtain individual cells
	<ol>
		<li>Cut the cryopreserved/thawed ovarian cortex tissue into small pieces of approximately 1 mm x 1 mm x 1 mm using a scalpel.</li>
		<li>Enzymatically digest the tissue fragments in 4 mL of pre-warmed (37 &#176;C) L15 medium containing 0.1 mg/mL tissue dissociation enzyme mix, 10 &#181;g/mL DNase I, and 1 mg/mL collagenase I from C. histolyticum for a maximum of 75 min at 37 &#176;C. Pipet the digestion mix up and down every 15 min.</li>
		<li>Stop the enzymatic digestion by adding 4 mL of cold L15 supplemented with 10% fetal bovine serum (FBS). Wash the dissociated tissue once with 8 mL of cold L15 medium by centrifugation at 500 x g and resuspend in 500 &#181;L of L15 medium without vortexing to avoid damage to the cells.</li>
		<li>Transfer the cell suspension containing largely individual stromal cells and small follicles (oocytes surrounded by a single layer of granulosa cells) to a plastic Petri dish and examine the cell suspension under a stereomicroscope (100x magnification).</li>
		<li>Pick up the small follicles (&#60;50 &#181;m) manually by using a 75 &#181;m plastic pipette and transfer the follicles to a droplet of L15 medium supplemented with 10% FBS at 4 &#176;C to prevent the aggregation of follicles. Perform follicle pick-up for a maximum of 30 min. To improve follicular cell spreading prior to FISH analysis, transfer the purified follicles to a solution of 0.06% trypsin, 1 mg/mL ethylenediaminetetraacetic acid (EDTA), and 1 mg/mL glucose and incubate for 20 min at 37 &#176;C.</li>
		<li>Obtain ovarian stromal cells from the cortex cell suspension using a 75 &#181;m plastic pipette, taking special care to avoid contamination with small follicles.</li>
	</ol>
	</li>
	<li>FISH analysis of individual ovarian cell
	<ol>
		<li>Transfer the treated ovarian follicles (recommended n = 5-20) with trypsin/EDTA/glucose or stromal cells (n &#62; 1,000) to droplets of 5 &#181;L of 0.15 mM KCl/15 &#181;L of Dulbecco&#39;s phosphate buffered saline (DPBS) on a slide and incubate for 20 min at 37 &#176;C.</li>
		<li>Dry and pre-fixate the slides in 300 &#181;L of 0.05 mM KCl/7.5% acetic acid/22.5% methanol for 2 min at room temperature (RT). Cover the slides with methanol/acetic acid (3:1) for 2 min at RT to finalize fixation.</li>
		<li>Make 20x standard sodium citrate (SSC) by adding 876 g of sodium chloride and 441 g of tri-sodium citrate dihydrate in 5 L of distilled water. Next, add 100 mL of the 20x SSC to 900 mL of demineralized (demi) water to obtain 2x SSC. Wash the sample in 2x SSC at 73 &#176;C, cover it with 100 &#181;L of 2% proteinase K, and seal it with a coverslip. Incubate the slides for 10 min at 37 &#176;C in the hybridization station.</li>
		<li>Remove the coverslip and wash the slides for 5 min in DPBS at RT. Fixate the sample for 5 min with 1% formaldehyde at RT. At this stage, the material is not yet fully attached to the glass slides and should therefore not be placed on a shaking platform.</li>
		<li>Wash the slides for 5 min in DPBS at RT, followed by dehydration in subsequent 70%, 80%, 90%, and 100% ethanol for 2 min each. Air-dry the dehydrated sample and hybridize it with fluorescently labeled probes.</li>
		<li>Select a centromere-specific probe for chromosome X and another chromosome-specific centromeric probe as a control to determine the X chromosomal content of ovarian cells. In this case, centromere-specific probes for chromosome X (CEP X [DXZ1]) directly labeled with fluorochrome SpectrumGreen and chromosome 18 (CEP 18 [D18Z1]) directly labeled with fluorochrome SpectrumOrange are used.</li>
		<li>Add 1 &#181;L of CEP X, 1 &#181;L of CEP 18, and 18 &#181;L of the hybridization buffer to the sample and seal with a coverslip that is glued to the slides to prevent evaporation of the probe during hybridization. Transfer the slides to the hybridization station for denaturation at 73 &#176;C for 3 min, followed by hybridization during an overnight incubation at 37 &#176;C.</li>
		<li>Remove the coverslip and any remaining glue from the slide after hybridization. Wash the slides in 0.4x SSC/0.3% Tween-20 at 72 &#176;C for 2 min, followed by incubation for 1 min in 2x SSC/0.1% Tween-20 at RT.</li>
		<li>Dehydrate the slides by subsequent 2 min incubations in 70%, 80%, 90%, and 100% ethanol and air-dry in the dark. Cover the slides with a mounting medium containing 4&#8242;,6-diamidino-2-phenylindole (DAPI). Keep at -20 &#176;C for at least 10 min before analysis by fluorescence microscopy.</li>
	</ol>
	</li>
	<li>Imaging
	<ol>
		<li>Examine the signal(s) for the X chromosome with a fluorescence microscope linked to an image processing software.
		<ol>
			<li>First, select fluorochrome DAPI.
			<ol>
				<li>Acquire an image by selecting New Cell &#62; Live &#62; Capture at 630x magnification. A new window with the threshold will appear. Set the blue bar of the threshold to 0 to minimize background and the red bar to maximum (255) to make the signals brighter. Click on Accept.</li>
				<li>A new window with enhancement will appear with a suggestion for darker/brighter (12), radius (3), and depth (1). Use the suggested values or adjust them if not satisfied.</li>
			</ol>
			</li>
			<li>Secondly, select fluorochrome SpectrumOrange and click on Capture.
			<ol>
				<li>Set the blue bar of the threshold to 0 to minimize background and the red bar to maximum (255) to make the signals brighter. Click on Accept.</li>
				<li>The window with enhancement will appear with a suggestion for darker/brighter (-11), radius (2.7), and depth (0.6). Use the suggested values or adjust them if not satisfied.</li>
			</ol>
			</li>
			<li>Finally, select fluorochrome SpectrumGreen and click on Capture.
			<ol>
				<li>Set the blue bar of the threshold to 0 to minimize background and the red bar to maximum (255) to make the signals brighter. Click on Accept.</li>
				<li>The window with enhancement will appear with a suggestion for darker/brighter (0), radius (3), and depth (0). Use the suggested values or adjust it if not satisfied. Save the image in a newly created file. 
				NOTE: Somatic cells were only evaluated when two signals of the control chromosome 18 were visible. In most oocytes, only one signal could be detected for each chromosome.</li>
			</ol>
			</li>
		</ol>
		</li>
		<li>Store the slides in the dark at 4 &#176;C after analysis to prevent loss of signals.</li>
	</ol>
	</li>
</ol>
2. FISH on paraffin sections of grafted ovarian cortex tissue
NOTE: One fragment of cryopreserved/thawed ovarian cortex tissue of 18 females with TS was xenografted into severe combined immunodeficient (SCID) mice for 5 months. The procedure of xenografting has been described previously and was conducted at the Universit&#233; Catholique de Louvain (Brussels, Belgium) following the local guidelines of the Committee on Animal Research regarding animal welfare (reference 2014/UCL/MD/007)18,19.
<ol>
	<li>Selecting sections of xenografted ovarian cortex tissue containing follicles
	<ol>
		<li>Fixate xenografted ovarian cortex tissue in 4% formaldehyde and embed the tissue in paraffin. Trim the blocks with a scalpel to remove extra paraffin and cut the paraffin block to 4 &#181;m thickness on a rotation microtome.</li>
		<li>Select every seventh section of the paraffin ribbon for hematoxylin and eosin (HE) staining to determine which sections contain follicles. Put the section in a water bath at 40-45 &#176;C and mount them on immunohistochemistry microscope slides.</li>
	</ol>
	</li>
	<li>Deparaffinization and HE staining
	<ol>
		<li>Put the slides on a stove for 10 min at 60 &#176;C, and thereafter immerse the slides in 100% xylene for 5 min. It is not necessary to place the slides directly on the stove. Hydrate the sections for 15 s in 100% ethanol, followed by 2 x 15 s in 96% ethanol. Rinse the slides in tap water for 2 min.</li>
		<li>Stain the slides in hematoxylin for 10 min and thereafter rinse the slides briefly in tap water. Briefly immerse the slides in a bicarbonate solution (100 g of magnesium sulfate and 10 g of sodium bicarbonate in 5 L of distilled water). Rinse the slides in tap water for 5 min.</li>
		<li>Counterstain the slides with eosin for 4 min and dehydrate the slides three times with 100% ethanol, followed by xylene. Coverslip the HE stains on the slides and evaluate the HE sections under a light microscope to select the sections with follicles (100x magnification).</li>
	</ol>
	</li>
	<li>Pre-treatment and hybridization of paraffin sections for DNA FISH
	<ol>
		<li>Select new sections that lay before or after the section that contained follicles from the paraffin ribbon. Mount one section on a glass slide. Dry the paraffin sections for at least 45 min on a stove at 56 &#176;C. It is not necessary to place the slides directly on the stove.</li>
		<li>Deparaffinize the sections in xylene for 10 min. Immerse the slides in 99.5% ethanol and rinse them for 5 min in tap water. Pre-treat the slides with target retrieval solution (low pH) for 10 min at 96 &#176;C. After cooling down, rinse the slides in distilled water.</li>
		<li>Treat the slides for 5 min with 0.01 M hydrochloric acid, followed by pepsin digestion (200 U/mL) for 15 min at 37 &#176;C. Rinse the slides again in 0.01 M hydrochloric acid and subsequently in PBS.</li>
		<li>Fixate the slides in 1% formaldehyde/PBS for 5 min. Rinse the slides briefly in PBS, and then again in demi water. Dehydrate the slides in 99.5% ethanol and let them air-dry.</li>
		<li>Select a centromere-specific probe for chromosome X and another chromosome-specific centromeric probe as a control to determine the X chromosomal content of granulosa cells. Here, chromosome 18 is used as a control.</li>
		<li>Apply 5 &#181;L of probe CEP 18 (D18Z1) directly labeled with fluorochrome SpectrumGreen and probe CEP X (DXZ1) directly labeled with fluorochrome SpectrumRed on the pre-treated slides. Apply a coverslip and seal the area with photo glue. Place the slides in a hybridizer for denaturation at 80 &#176;C for 10 min and hybridization overnight at 37 &#176;C.</li>
		<li>The next day, rinse the slides for 5 min in 2x SSC at 42 &#176;C, followed by a 2 min and a 1 min rinse in 0.3% Nonidet P40 at 73 &#176;C. Refresh the 2x SSC and rinse the slides again for 5 min at room temperature. Cover the cuvette so that the sections are kept in the dark.</li>
		<li>Rinse the slides briefly in distilled water. Dehydrate the slides in 99.5% ethanol and let them air-dry again. Finally, mount the slides with a solution containing DAPI and mounting medium.</li>
	</ol>
	</li>
	<li>Imaging
	<ol>
		<li>Analyze the results under a fluorescence microscope at 630x magnification. Open the image processing software on the computer. Select FISH as the profile.</li>
		<li>Check if the DAPI emission is set at 431 nm and the excitation at 359 nm, Texas red emission at 613 nm and excitation at 595 nm, and FITC emission at 519 nm and excitation at 495 nm.</li>
		<li>Acquire an image by selecting Live &#62; Capture Single Image. Optimize the image quality by adjusting the exposure and gain by moving the Exposure Slider and Gain Slider in the Image menu on the left (e.g., exposure: 212 ms, and gain: 7.9). The required exposure and gain can vary per image; observe the changes during this process to obtain an optimized image. Save the image in a newly created file.</li>
		<li>Store the slides in the dark at 4 &#176;C after analysis to prevent loss of signals.</li>
	</ol>
	</li>
</ol>

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<li>Huber, D., von Voithenberg, L. V., Kaigala, G. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fluorescence+in+situ+hybridization+%28FISH%29%3A+History%2C+limitations+and+what+to+expect+from+micro-scale+FISH%5BTitle%5D)+AND+%22Micro+and+Nano+Engineering%22%5BJournal%5D)">Fluorescence in situ hybridization (FISH): History, limitations and what to expect from micro-scale FISH.</a> Micro and Nano Engineering. 1, 15-24 (2018).</li>
<li>Hu, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fluorescence+in+situ+hybridization+%28FISH%29%3A+an+increasingly+demanded+tool+for+biomarker+research+and+personalized+medicine%5BTitle%5D)+AND+%22Biomarker+Research%22%5BJournal%5D)">Fluorescence in situ hybridization (FISH): an increasingly demanded tool for biomarker research and personalized medicine.</a> Biomarker Research. 2 (1), 3(2014).</li>
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</ol>

The authors have nothing to disclose.

FISH analysis is a well-known technique to detect X chromosomal aberrations in lymphocytes or buccal cells of both males and females10. Several studies have described FISH on gametes of males with X chromosomal aberrations, but detailed information obtained by FISH on ovarian cells from females with X chromosomal aberrations is still lacking14. This article presents novel methods based on FISH to determine if aneuploidy is present in the ovarian cells of non-grafted and grafted ovarian cortex tissue from females with X chromosomal aberrations.
The main challenge of the protocol for the isolation of individual ovarian cells in non-grafted ovarian cortex tissue lies in the enzymatic digestion of the tissue, which requires some practice beforehand. In order to obtain accurate FISH signals of sufficient ovarian cells, it is important to follow the steps regarding enzymatic digestion, incubation times, and indicated temperatures strictly. Deviating from the protocol can cause a substantial loss of ovarian cells during the process, and this should especially be avoided in patients who already have a low ovarian reserve. Another critical step in this method is to treat the purified primordial follicles with trypsin to prevent oocytes and the granulosa cell mass from clumping, which precludes the analysis of individual cells. Without treatment with trypsin, the number of individual granulosa cells that can be reliably analyzed by FISH is severely reduced. 
The FISH analysis on ovarian cortex tissue retrieved after long-term grafting requires a different technique, as only small follicles were found to be sufficiently susceptible to the enzymatic digestion that is necessary to obtain individual ovarian cells. In addition, similar to autotransplantation of ovarian cortex tissue, many follicles get lost after grafting due to hypoxia and a lack of nutrients before the graft is sufficiently revascularized by the host20. The number of follicles after grafting is therefore expected to be considerably lower than before grafting. For this FISH technique used for histological sections, it is important to fixate the grafted tissue in 4% formaldehyde only to obtain optimal FISH signals. Fixation of ovarian cortex tissue in Bouin's fixative is routinely used in the laboratory, and while this gives excellent results when combined with standard HE staining, fixating tissue with Bouin's prior to FISH leads to weak and blurry fluorescent signals that are difficult to interpret.
Although this protocol has been proven to be successful in determining the X chromosomal content of ovarian cells, it still has some limitations. One limitation is that these methods can only be used to analyze the ovarian cells of females with numerical aberrations. Numerical aberrations can be detected by using probes directed at repetitive sequences21. These probes hybridize multiple repeating base pair sequences in the centromere region, resulting in strong hybridization signals. In contrast, structural aberrations can only be detected by using probes against unique single sequences. These probes hybridize to sequences that only occur once in the haploid genome, resulting in a considerably less intense FISH signal compared to numerical aberrations. Due to these relatively weak FISH signals, it is difficult to properly determine the X chromosomal content of ovarian cells in females with structural aberrations. 
Secondly, FISH signals of oocytes of small follicles in non-grafted tissue are difficult to analyze, due to the close proximity of the four sister chromatids in the prophase of meiosis I22. Only one strong hybridization signal will be present in the oocytes, and therefore it is not possible to simply count the number of signals in the oocytes to determine the X chromosomal content. Instead, the surface ratio of chromosome X and 18 FISH signals should be used to determine the X chromosomal content in these cells. This can only be reliably determined if the FISH signals in the oocytes are clearly present. 
Furthermore, FISH on grafted tissue can only be used to determine the X chromosomal content of granulosa cells from secondary and antral follicles, as small follicles in histological sections of grafted tissue only have a few granulosa cells that can be properly analyzed. In addition, the X chromosomal content of oocytes cannot be determined accurately using this method due to the large diameter of the oocytes. 
Finally, it remains challenging to obtain female gametes compared to male gametes because invasive surgery is required to obtain ovarian cells or ovarian cortex tissue. Therefore, these methods are most likely to be applied in a research setting.
In conclusion, FISH analysis of ovarian cells of non-grafted and grafted ovarian cortex tissue from females with X chromosomal aberrations is a unique and useful technique to gain insight into the X chromosomal content of ovarian cells in this specific group. These techniques show that cryopreservation of ovarian cortex tissue from females with X chromosomal aberrations is possible, and that cryopreserved primordial follicles are able to grow to secondary and antral follicles. However, it should be kept in mind that both methods are intended to facilitate future research in females with X chromosomal aberrations, and are not designed to be used as a diagnostic tool to screen reproductive outcomes of females with X chromosomal aberrations in clinical practice.

Infertility is a health issue of the male or female reproductive system, affecting approximately 186 million individuals of reproductive age worldwide1. In at least 35% of infertile couples, infertility is caused by a disorder of the female reproductive system2. There are many factors that can cause female infertility, such as genetic factors, genital tract abnormalities, endocrine dysfunction, inflammatory diseases, and iatrogenic treatment3.
Genetic abnormalities are present in approximately 10% of infertile females4,5. Of all genetic abnormalities, X chromosome aberrations are the most common cause of ovarian dysgenesis2. Several studies have reported that X chromosomal aberrations in females with Turner syndrome (TS) or Triple X syndrome are associated with premature ovarian failure due to an accelerated loss of germ cells or impaired oogenesis6,7,8.
Aberrations of the X chromosome can be divided into: 1) numerical aberrations, in which the number of X chromosomes is different but the X chromosomes are intact; and 2) structural aberrations, in which the X chromosome has gained or lost genetic material3,9. Numerical aberrations of the X chromosome are more common than structural abnormalities and are often caused by spontaneous errors during cell division3,9. When such an error occurs during meiosis, it may lead to aneuploid gametes and ultimately to offspring with chromosomal aberrations in all cells. When chromosome defects arise in somatic cells as a result of errors occurring during mitosis in the early stages of ontogenesis, it may lead to mosaicism. In these individuals, both cells with normal X chromosomal content and cells with X chromosomal aberrations are present.
In the 1980s, a cytogenetic technique called fluorescence in situ hybridization (FISH) was developed to visualize and locate specific nucleic acid sequences on metaphase and interphase chromosomes10,11. This technique uses fluorescent-labeled DNA probes to bind to a specific sequence in the chromosome, which can then be visualized by using a fluorescence microscope.
Nowadays, FISH is widely used as a clinical diagnostic tool and is considered the gold standard in detecting chromosomal aberrations10. In the field of reproductive medicine, FISH analysis on sperm has been used to gain insight into the X chromosomal content of spermatozoa in males with numerical or structural chromosomal aberrations in somatic cells12,13,14. These studies showed that males with chromosomal aberrations were more likely to have a higher frequency of aneuploid spermatozoa present in their semen compared to males with normal karyotypes12,13,14.
In contrast to spermatozoa, very little is known about the X chromosomal content of ovarian cells (including oocytes, granulosa/theca cells, and stromal cells) in individuals with a chromosomal aberration, as well as the possible consequences of aneuploidy of these cells on their reproductive potential. An important reason for the scarce information on the karyotype of ovarian cells compared to spermatozoa is the fact that women have to undergo an invasive procedure such as a follicle puncture or surgery to obtain oocytes or ovarian cortex tissue. Female gametes are, therefore, difficult to obtain for research purposes.
Currently, an observational intervention study is being performed in the Netherlands to explore the efficacy of ovarian tissue cryopreservation in young females with TS15. One fragment of the ovarian cortex tissue of the patient was available to identify the X chromosomal content of the ovarian cells16,17. As part of the study, a novel method was developed based on FISH of dissociated ovarian cortex tissue to determine if chromosomal aberrations are present in ovarian cells in females carrying a chromosomal aberration in non-ovarian somatic cells, such as lymphocytes or buccal cells. In addition, the effect of aneuploidy in ovarian cells on folliculogenesis was determined as well. To this end, an established FISH protocol was modified that enables the analysis of histological sections of ovarian cortex tissue after artificially induced folliculogenesis during long term xenotransplantation in immunocompromised mice. In this study, we present two methods based on FISH to determine the X chromosomal content in ovarian cells in non-grafted and grafted ovarian cortex tissue in females with X chromosomal aberrations, with the aim to improve basic science on this topic.

<table><tbody><tr><td>Acetic acid</td><td>Biosolve BV</td><td>0001070602BS</td><td></td></tr><tr><td>Centrifuge 1200</td><td>Hettich Universal</td><td>4140</td><td></td></tr><tr><td>Collagenase I</td><td>Sigma</td><td>131470</td><td></td></tr><tr><td>Coverslip</td><td>VWR</td><td>0631-0146</td><td></td></tr><tr><td>DAPI</td><td>Vector</td><td>H-1200</td><td></td></tr><tr><td>DNase I</td><td>Roche</td><td>10104159001</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Phosphate Buffered Saline&amp;nbsp;</td><td>Lonza</td><td>BE17-513Q</td><td></td></tr><tr><td>EDTA</td><td>Merck</td><td>108421</td><td></td></tr><tr><td>Eosin-Y</td><td>Sigma</td><td>1159350100</td><td></td></tr><tr><td>Ethanol</td><td>EMSURE</td><td>1009832500</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Life technology</td><td>10100147</td><td></td></tr><tr><td>Fluorescence microscope for sections DM4 B</td><td>Leica Microsystems&amp;nbsp;</td><td></td><td></td></tr><tr><td>Fluorescence microscope scope A1</td><td>Zeiss AXIO</td><td></td><td></td></tr><tr><td>Fluorescent labeled probes for dissociated cells</td><td>Abbott Diagnostics</td><td>CEPX (DXZ1) 05J1023&lt;br/&gt; CEP18 (D18Z1) 05J0818</td><td></td></tr><tr><td>Fluorescent labeled probes for tissue sections</td><td>Abbott Diagnostics</td><td>CEP X (DXZ1 05J08-023&lt;br/&gt; CEP 18 (D18Z1)&amp;nbsp; 05J10-028</td><td></td></tr><tr><td>Formaldehyde</td><td>Sigma</td><td>252549</td><td></td></tr><tr><td>Glucose</td><td>Merck</td><td>108337</td><td></td></tr><tr><td>Glue (Fixogum)</td><td>Leica Microsystems</td><td>LK071A</td><td></td></tr><tr><td>Hematoxylin</td><td>Sigma</td><td>1159380025</td><td></td></tr><tr><td>Hybridization buffer</td><td>Abott Diagnostics</td><td>32-804826/06J67-001</td><td></td></tr><tr><td>Hybridization Station&amp;nbsp;</td><td>Dako</td><td>S2451</td><td></td></tr><tr><td>Hydrochloric acid</td><td>Merck</td><td>1003171000</td><td></td></tr><tr><td>Image processing software individual ovarian cortex cells (Cytovision 7.7)</td><td>Leica Biosystems</td><td></td><td></td></tr><tr><td>Image processing software on paraffine sections&amp;nbsp;</td><td>Leica Application Suitex (3.7.5.24914)</td><td></td><td></td></tr><tr><td>Immunohitochemistry microscope slides</td><td>Dako</td><td>K802021-2</td><td></td></tr><tr><td>L15</td><td>Lonza</td><td>12-700Q</td><td></td></tr><tr><td>Liberase DH</td><td>Roche</td><td>05 401 151 001</td><td></td></tr><tr><td>Light microscope</td><td>Zeiss West Germany</td><td></td><td></td></tr><tr><td>Magnesium sulphate</td><td>Merck</td><td>A335586</td><td></td></tr><tr><td>Methanol</td><td>Honeywell</td><td>14262-1L</td><td></td></tr><tr><td>Mounting medium</td><td>Vectashield, Vector</td><td>H-1000</td><td></td></tr><tr><td>Nonidet P40</td><td>Sigma</td><td>7385-1L</td><td></td></tr><tr><td>Paraffin</td><td>Poth Hile</td><td>2712.20.10</td><td></td></tr><tr><td>Pepsin</td><td>Sigma</td><td>P7000-25G</td><td></td></tr><tr><td>Phosphate-Buffered Saline (PBS)</td><td>Gibco</td><td>11530546</td><td></td></tr><tr><td>Plastic pipette</td><td>CooperSurgical</td><td>7-72-4075/1</td><td></td></tr><tr><td>Potassium chloride&amp;nbsp;</td><td>Merck</td><td>1049361000</td><td></td></tr><tr><td>Proteinase K</td><td>Qiagen</td><td>19131</td><td></td></tr><tr><td>Rotation microtome HM 355S</td><td>Thermo sceintific</td><td></td><td></td></tr><tr><td>Scalpel</td><td>Dahlhausen</td><td>11.000.00.515</td><td></td></tr><tr><td>Slide for FISH on dissociated cells</td><td>Thermo scientific</td><td>J1810AM1JZ</td><td></td></tr><tr><td>Sodium bicarbonate</td><td>Sigma</td><td>55761-500G</td><td></td></tr><tr><td>Standard Sodium Citrate (SSC)</td><td>Fisher Scientific, Invitrogen</td><td>10515203</td><td></td></tr><tr><td>Stereomicroscope IX 70</td><td>Olympus</td><td></td><td></td></tr><tr><td>Target Retrieval Solution &amp;nbsp;&amp;nbsp;</td><td>Dako</td><td>GV80511-2</td><td></td></tr><tr><td>Trypsin</td><td>Sigma</td><td>T4799</td><td></td></tr><tr><td>Tween-20</td><td>ThermoFisher</td><td>85113</td><td></td></tr><tr><td>Xylene</td><td>BOOM</td><td>760518191000</td><td></td></tr></tbody></table>

exploring x chromosomal aberrations in ovarian cells by using fluorescence in situ hybridization

Millions of people worldwide deal with issues concerning fertility. Reduced fertility, or even infertility, may be due to many different causes, including genetic disorders, of which chromosomal abnormalities are the most common. Fluorescence in situ hybridization (FISH) is a well-known and frequently used method to detect chromosomal aberrations in humans. FISH is mainly used for the analysis of chromosomal abnormalities in the spermatozoa of males with numerical or structural chromosomal aberrations. Furthermore, this technique is also frequently applied in females to detect X chromosomal aberrations that are known to cause ovarian dysgenesis. However, information on the X chromosomal content of ovarian cells from females with X chromosomal aberrations in lymphocytes and/or buccal cells is still lacking.
The aim of this study is to advance basic research regarding X chromosomal aberrations in females, by presenting two methods based on FISH to identify the X chromosomal content of ovarian cells. First, a method is described to determine the X chromosomal content of isolated ovarian cells (oocytes, granulosa cells, and stromal cells) in non-grafted ovarian cortex tissue from females with X chromosomal aberrations. The second method is directed at evaluating the effect of chromosomal aberrations on folliculogenesis by determining the X chromosomal content of ovarian cells of newly formed secondary and antral follicles in ovarian tissue, from females with X chromosomal aberrations after long-term grafting into immunocompromised mice. Both methods could be helpful in future research to gain insight into the reproductive potential of females with X chromosomal aberrations.

FISH on isolated ovarian cells prior to grafting 
Cryopreserved ovarian cortex tissue from females with 45,X/46,XX (patient A) or 45,X/46,XX/47,XXX (patient B) TS were used to illustrate the results using this protocol. In patient A, 50% of the lymphocytes had a 45,X karyotype and 50% had 46,XX. In patient B, 38% of the lymphocytes were 45,X, 28% were 46,XX, and 34% were 47,XXX. Centromere-specific probes for chromosome X (green) and chromosome 18 as the control (red) were used to determine the X chromosomal content of individual granulosa cells, stromal cells, and oocytes isolated from ovarian cortex tissue of TS patients without prior xenotransplantation (Figure 1).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64734/64734fig01.jpg" /> 
Figure 1: FISH analysis of isolated ovarian cells from ovarian cortex tissue prior to grafting. Oocytes (arrow heads) and granulosa cells from single primordial follicles&#160;(A,B)&#160;and (C,D) the surrounding stromal cells were analyzed with fluorescent specific probes for chromosome X (green signals) and control chromosome 18 (red signals). White arrows indicate 45,X cell lines, yellow arrows indicate 46,XX cell lines, and red arrows indicate 47,XXX cell lines. Not all fluorescent signals are in the same plane of focus. Bars represent 10 &#181;m. The magnification of FISH signals was set at 630x. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64734/64734fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The differences between individual primordial follicles that were treated with and without trypsin before FISH are shown in Figure 2. By using trypsin prior to the FISH analysis, the granulosa cell mass and oocytes were less clumped, allowing the analysis of the X chromosomal content of individual granulosa cells and oocytes. The DNA of oocytes can easily be distinguished from that of the surrounding granulosa cells due to the irregular shape, size, and diffuse appearance of DNA from the oocytes. In addition, only one strong FISH signal for each chromosome is observed in the oocytes of small follicles, due to the close proximity of the four sister chromatids in these cells. The X chromosomal content of oocytes can be determined by using the surface ratio of the FISH signal for chromosome X to that of chromosome 18.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64734/64734fig02.jpg" /> 
Figure 2: Small follicles treated with and without trypsin prior to FISH. (A)&#160;Enzymatic digestion of ovarian cortex tissue resulted in a suspension of largely dissociated cells but leaving the primordial follicles, consisting of an intact oocyte surrounded by a single layer of granulosa cells (arrow heads in panel A). (B) Small follicles were handpicked from the cell suspension but provided difficulties to interpret signals after FISH due to clumping of the granulosa cells&#160;(C). (D,E) Additional digestion of the isolated follicles with trypsin prior to FISH resulted in individual granulosa cells to contract into a spherical morphology on the surface of the follicles and were more likely to dissociate from the follicle to become accessible for FISH analysis. Oocyte-derived FISH signals are indicated by arrows. Black bars represent 100 &#181;m and white bars represent 10 &#181;m. The magnification of FISH signals was set at 630x. Panel D has been reproduced with permission from Peek&#160;et al.16. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64734/64734fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
FISH analysis of granulosa cells on paraffin sections of grafted ovarian cortex tissue 
Secondary and antral follicles were found to be less susceptible to enzymatic digestion, which makes the previously described method of tissue dissociation to obtain individual ovarian cells not suitable for growing follicles found in the ovarian tissue after long term xenografting. Therefore, the FISH protocol was optimized using 4 &#181;m histological sections to determine the X chromosomal content of granulosa cells of secondary and antral follicles in ovarian cortex tissue after grafting (Figure 3). In this setting, it is unlikely that the FISH signal of either the X chromosome or the control chromosome in oocytes would be captured in a single 4 &#181;m section, due to the large diameter of oocytes in the growing follicles.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64734/64734fig03.jpg" /> 
Figure 3: Histological and FISH staining of ovarian cortex tissue sections after xenotransplantation. (A,B)&#160;Hematoxylin/eosin staining showed morphologically normal secondary and antral follicles in ovarian cortex tissue after 5 months of xenografting. (C,D) The X chromosomal content of granulosa cells of these follicles was determined by FISH analysis. In this figure, chromosome X is shown in red and chromosome 18 in green. The bar in panel A represents 50 &#181;m, the bar in panel B represents 20 &#181;m, and the bars in panels C and D represent 10 &#181;m. The magnification of FISH signals was set at 630x. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64734/64734fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
As a first step, experiments with different fixating techniques of the tissues were performed to determine which method results in the best FISH signals. FISH analysis was performed on grafted tissue that was fixated in both Bouin&#39;s solution and subsequently immersed in 4% formaldehyde, and on grafted tissue that was fixated in 4% formaldehyde only. Grafted tissue that was first fixated in Bouin&#39;s displayed a green haze on the image, making it difficult to accurately count the number of FISH signals (Figure 4).
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64734/64734fig04.jpg" /> 
Figure 4: Grafted ovarian cortex tissue fixated in Bouin&#39;s solution and formaldehyde only. (A)&#160;Fixating grafted ovarian cortex tissue in Bouin&#39;s solution resulted in blurred and largely obscured fluorescent signals in follicular cells due to a green haze. (B) Ovarian cortex tissue that was fixated in formaldehyde only provided excellent fluorescent signals that were easy to interpret. Bars represent 10 &#181;m. The magnification of FISH signals was set at 630x. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64734/64734fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
In order to determine the X chromosomal content of granulosa cells of follicles in histological sections, it is not possible to simply count the FISH signals of granulosa cells. Part of the chromosomes of an individual granulosa cell may be lost in a certain histological section, due to sectioning of the tissue. Therefore, it is necessary to first determine the percentage of FISH signals lost due to sectioning before ultimately determining the loss of X chromosomal content of the granulosa cell population due to aneuploidy in newly formed secondary and antral follicles after grafting of the tissue.
The percentage of lost FISH signals due to sectioning can be calculated by determining the number of FISH signals per granulosa cell of the non-aneuploid control chromosome 18. It is expected that the same percentage of the X chromosome is lost due to sectioning. Any additional reduction in the number of X chromosome FISH signals in the granulosa cell population is due to aneuploidy. However, using FISH signals of a control chromosome to determine X chromosomal loss due to aneuploidy requires the detection sensitivity of the FISH signals of the X chromosome and the control chromosome to be very similar. The detection sensitivity of both FISH probes in granulosa cells of growing follicles in histological sections of non-aneuploid 46,XX individuals was therefore determined. When the ratio of chromosome X/control chromosome FISH signals is close to 1, it can be assumed that the detection sensitivity of both probes are indeed very similar, and that the FISH probes can thus be used to determine the level of aneuploidy in the granulosa cell population of growing follicles in histological sections of ovarian tissue after xenotransplantation.
Example 
When analyzing the number of chromosome 18 signals in 130 granulosa cells of a follicle from an ovary of a 46,XX individual, it is expected that 260 signals are present. However, in a 4 &#181;m section of these cells, only 204 chromosome 18 signals were observed. This indicates that 21.5% of the signals are lost due to sectioning ((260-204)/260 x 100 = 21.5%). The number of chromosome 18 signals per granulosa cell is therefore reduced to 204/130 = 1.57 due to sectioning.
Next, the number of X chromosomes in granulosa cells of an antral follicle after grafting in ovarian tissue of a female with TS is analyzed. In total, 191 signals for chromosome X and 199 signals for chromosome 18 were counted. The number of granulosa cells can be determined by dividing the total number of signals for chromosome 18 by the number of chromosome 18 signals per granulosa cell (199/1.57 = 127 granulosa cells). Finally, the percentage of 45,X granulosa cells can be determined by dividing the difference in chromosome 18 and chromosome X signals with the number of granulosa cells (i.e., [199-191]/127 x 100 = 6% of the granulosa cells are 45,X, and 94% of these cells are 46, XX).
It is noteworthy that, in patients with a 47,XXX cell line, it is only possible to determine the minimum percentage of granulosa cells with a 47,XXX karyotype, since a mix of 45,X and 47,XXX granulosa cells has the same number of X chromosome signals as 46,XX granulosa cells.

Watch this Scientific Journal Video about Exploring X Chromosomal Aberrations in Ovarian Cells by Using Fluorescence In Situ Hybridization at JoVE.com

Exploring X Chromosomal Aberrations in Ovarian Cells by Using Fluorescence In Situ Hybridization

This article presents two methods based on fluorescence in situ hybridization to determine the X chromosomal content of ovarian cells in non-grafted and grafted ovarian cortex tissue from females with X chromosomal aberrations.

Exploring X Chromosomal Aberrations in Ovarian Cells by Using Fluorescence In Situ Hybridization

Millions of people worldwide deal with issues concerning fertility. Reduced fertility, or even infertility, may be due to many different causes, including ...

exploring-x-chromosomal-aberrations-ovarian-cells-using-fluorescence

Nanyang Technological University

Research

JoVE Journal

Genetics

1.0K Views.  Radboudumc. FISH-analyse van eierstokcellen van vrouwtjes met X-chromosomale afwijkingen is een unieke techniek om inzicht te krijgen in het X-chromosomale gehalte van eierstokcellen in deze specifieke groep. Deze technieken kunnen nuttig zijn om meer te weten te komen over het voortplantingspotentieel bij vrouwen met X-chromosomale afwijkingen. Het is belangrijk om te vermelden dat beide methoden bedoeld zijn om toekomstig onderzoek te vergemakkelijken en niet zijn ontworpen om te worden gebruikt als ee...

Video: Exploring X Chromosomal Aberrations in Ovarian Cells by Using Fluorescence In Situ Hybridization

Detection of Inter-chromosomal Stable Aberrations by Multiple Fluorescence In Situ Hybridization (mFISH) and Spectral Karyotyping (SKY) in Irradiated Mice

Ionizing radiation (IR) induces numerous stable and unstable chromosomal aberrations. Unstable aberrations, where chromosome morphology is substantially compromised, can easily be identified by conventional chromosome staining techniques. However, detection of stable aberrations, which involve exchange or translocation of genetic materials without considerable modification in the chromosome morphology, requires sophisticated chromosome painting techniques that rely on in situ hybridization of fluorescently labeled DNA probes, a chromosome painting technique popularly known as fluorescence in situ hybridization (FISH). FISH probes can be specific for whole chromosome/s or precise sub-region on chromosome/s. The method not only allows visualization of stable aberrations, but it can also allow detection of the chromosome/s or specific DNA sequence/s involved in a particular aberration formation. A variety of chromosome painting techniques are available in cytogenetics; here two highly sensitive methods, multiple fluorescence in situ hybridization (mFISH) and spectral karyotyping (SKY), are discussed to identify inter-chromosomal stable aberrations that form in the bone marrow cells of mice after exposure to total body irradiation. Although both techniques rely on fluorescent labeled DNA probes, the method of detection and the process of image acquisition of the fluorescent signals are different. These two techniques have been used in various research areas, such as radiation biology, cancer cytogenetics, retrospective radiation biodosimetry, clinical cytogenetics, evolutionary cytogenetics, and comparative cytogenetics.

Detection of Inter-chromosomal Stable Aberrations by Multiple Fluorescence In Situ Hybridization mFISH and Spectral Karyotyping SKY in Irradiated Mice

The present protocol describes the usefulness of multiple fluorescence in situ hybridization (mFISH) and spectral karyotyping (SKY) in identifying inter-chromosomal stable aberrations in the bone marrow cells of mice after exposure to total body irradiation.

Ionizing radiation (IR) induces numerous stable and unstable chromosomal aberrations. Unstable aberrations, where chromosome morphology is substantially ...

Using Fluorescence In Situ Hybridization (FISH) to Monitor the State of Arm Cohesion in Prometaphase and Metaphase I Drosophila Oocytes

In humans, chromosome segregation errors in oocytes are responsible for the majority of miscarriages and birth defects. Moreover, as women age, their risk of conceiving an aneuploid fetus increases dramatically and this phenomenon is known as the maternal age effect. One requirement for accurate chromosome segregation during the meiotic divisions is maintenance of sister chromatid cohesion during the extended prophase period that oocytes experience. Cytological evidence in both humans and model organisms suggests that meiotic cohesion deteriorates during the aging process. In addition, segregation errors in human oocytes are most prevalent during meiosis I, consistent with premature loss of arm cohesion. The use of model organisms is critical for unraveling the mechanisms that underlie age-dependent loss of cohesion. Drosophila melanogaster offers several advantages for studying the regulation of meiotic cohesion in oocytes. However, until recently, only genetic tests were available to assay for loss of arm cohesion in oocytes of different genotypes or under different experimental conditions. Here, a detailed protocol is provided for using fluorescence in situ hybridization (FISH) to directly visualize defects in arm cohesion in prometaphase I and metaphase I arrested Drosophila oocytes. By generating a FISH probe that hybridizes to the distal arm of the X chromosome and collecting confocal Z stacks, a researcher can visualize the number of individual FISH signals in three dimensions and determine whether sister chromatid arms are separated. The procedure outlined makes it possible to quantify arm cohesion defects in hundreds of Drosophila oocytes. As such, this method provides an important tool for investigating the mechanisms that contribute to cohesion maintenance as well as the factors that lead to its demise during the aging process.

Using Fluorescence In Situ Hybridization FISH to Monitor the State of Arm Cohesion in Prometaphase and Metaphase I Drosophila Oocytes

This manuscript presents a detailed method for generating X-chromosome arm probes and performing fluorescence in situ hybridization (FISH) to examine the state of sister chromatid cohesion in prometaphase and metaphase I arrested Drosophila oocytes. This protocol is suitable for determining whether meiotic arm cohesion is intact or disrupted in different genotypes.

In humans, chromosome segregation errors in oocytes are responsible for the majority of miscarriages and birth defects. Moreover, as women age, their risk ...

Developmental Biology

Fluorescence in situ hybridization (FISH) Protocol in Human Sperm

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic process in the parents. In human males, these errors can lead to the production of spermatozoa with numerical chromosome abnormalities which represent an increased risk of transmitting these anomalies to the offspring.
For this reason, the technique of fluorescence in situ hybridization (FISH) on sperm nuclei has become a protocol widely incorporated in the context of clinical diagnosis. This practice provides an estimate of the frequencies of numerical chromosome abnormalities in the gametes of the patients that seek for genetic reproductive advice.
To date, the chromosomes most frequently included in sperm FISH analysis are chromosomes X, Y, 13, 18 and 21.
This video-article describes, step by step, how to process and fix a human semen sample, how to decondense and denature the sperm chromatin, how to proceed to obtain sperm FISH preparations, and how to visualize the results at the microscope. Special remarks of the most relevant steps are given to achieve the best results.

Fluorescence in situ hybridization FISH Protocol in Human Sperm

This video-article describes, step by step, how to process a semen sample to achieve good-quality fluorescence in situ hybridization on human spermatozoa. Preparations obtained can be used for aneuploidy screening in the context of clinical diagnosis.

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic ...

Biology

Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency and type of chromosome aberrations in different cell types allows defects in DNA repair pathways to be elucidated. Understanding mammalian DNA repair biology has been greatly helped by the production of mice with knockouts in specific genes. The goal of this protocol is to quantify genomic instability in mouse B lymphocytes. Labeling of the telomeres using PNA-FISH probes (peptide nucleic acid - fluorescent in situ hybridization) facilitates the rapid analysis of genomic instability in metaphase chromosome spreads. B cells have specific advantages relative to fibroblasts, because they have normal ploidy and a higher mitotic index. Short-term culture of B cells therefore enables precise measurement of genomic instability in a primary cell population which is likely to have fewer secondary genetic mutations than what is typically found in transformed fibroblasts or patient cell lines.

DNA repair pathways are essential for maintenance of genomic integrity and preventing mutation and cancer. The goal of this protocol is to quantify genomic instability by direct observation of chromosome aberrations in metaphase spreads from mouse B cells using fluorescent in situ hybridization (FISH) for telomeric DNA repeats.

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency ...

Immunology and Infection

Quantifying Replication Stress in Ovarian Cancer Cells Using Single-Stranded DNA Immunofluorescence

Replication stress is a hallmark of several ovarian cancers. Replication stress can emerge from multiple sources, including double-strand breaks, transcription-replication conflicts, or amplified oncogenes, inevitably resulting in the generation of single-stranded DNA (ssDNA). Quantifying ssDNA, therefore, presents an opportunity to assess the level of replication stress in different cell types and under various DNA-damaging conditions or treatments. Emerging evidence also suggests that ssDNA can be a predictor of responses to chemotherapeutic drugs that target DNA repair. Here, we describe a detailed immunofluorescence-based methodology to quantify ssDNA. This methodology involves labeling the genome with a thymidine analog, followed by the antibody-based detection of the analog at the chromatin under non-denaturing conditions. Stretches of ssDNA can be visualized as foci under a fluorescence microscope. The number and intensity of the foci directly co-relate with the level of ssDNA present in the nucleus. We also describe an automated pipeline to quantify the ssDNA signal. The method is rapid and reproducible. Furthermore, the simplicity of this methodology makes it amenable to high-throughput applications such as drug and genetic screens.

Here, we describe an immunofluorescence-based method to quantify the levels of single-stranded DNA in cells. This efficient and reproducible method can be utilized to examine replication stress, a common feature in several ovarian cancers. Additionally, this assay is compatible with an automated analysis pipeline, which further increases its efficiency.

Replication stress is a hallmark of several ovarian cancers. Replication stress can emerge from multiple sources, including double-strand breaks, ...

Cancer Research

Visualizing DNA Damage Repair Proteins in Patient-Derived Ovarian Cancer Organoids via Immunofluorescence Assays

Immunofluorescence is one of the most widely used techniques to visualize target antigens with high sensitivity and specificity, allowing for the accurate identification and localization of proteins, glycans, and small molecules. While this technique is well-established in two-dimensional (2D) cell culture, less is known about its use in three-dimensional (3D) cell models. Ovarian cancer organoids are 3D tumor models that recapitulate tumor cell clonal heterogeneity, the tumor microenvironment, and cell-cell and cell-matrix interactions. Thus, they are superior to cell lines for the evaluation of drug sensitivity and functional biomarkers. Therefore, the ability to utilize immunofluorescence on primary ovarian cancer organoids is extremely beneficial in understanding the biology of this cancer. The current study describes the technique of immunofluorescence to detect DNA damage repair proteins in high-grade serous patient-derived ovarian cancer organoids (PDOs). After exposing the PDOs to ionizing radiation, immunofluorescence is performed on intact organoids to evaluate nuclear proteins as foci. Images are collected using z-stack imaging on confocal microscopy and analyzed using automated foci counting software. The described methods allow for the analysis of temporal and special recruitment of DNA damage repair proteins and colocalization of these proteins with cell-cycle markers.

Visualizing DNA Damage Repair Proteins in Patient-Derived Ovarian Cancer Organoids via Immunofluorescence Assays

The present protocol describes methods for evaluating DNA damage repair proteins in patient-derived ovarian cancer organoids. Included here are comprehensive plating and staining methods, as well as detailed, objective quantification procedures.

Immunofluorescence is one of the most widely used techniques to visualize target antigens with high sensitivity and specificity, allowing for the accurate ...

Chromosomics: Detection of Numerical and Structural Alterations in All 24 Human Chromosomes Simultaneously Using a Novel OctoChrome FISH Assay

Fluorescence in situ hybridization (FISH) is a technique that allows specific DNA sequences to be detected on metaphase or interphase chromosomes in cell nuclei1. The technique uses DNA probes with unique sequences that hybridize to whole chromosomes or specific chromosomal regions, and serves as a powerful adjunct to classic cytogenetics. For instance, many earlier studies reported the frequent detection of increased chromosome aberrations in leukemia patients related with benzene exposure, benzene-poisoning patients, and healthy workers exposed to benzene, using classic cytogenetic analysis2. Using FISH, leukemia-specific chromosomal alterations have been observed to be elevated in apparently healthy workers exposed to benzene3-6, indicating the critical roles of cytogentic changes in benzene-induced leukemogenesis. 
Generally, a single FISH assay examines only one or a few whole chromosomes or specific loci per slide, so multiple hybridizations need to be conducted on multiple slides to cover all of the human chromosomes. Spectral karyotyping (SKY) allows visualization of the whole genome simultaneously, but the requirement for special software and equipment limits its application7. Here, we describe a novel FISH assay, OctoChrome-FISH, which can be applied for Chromosomics, which we define here as the simultaneous analysis of all 24 human chromosomes on one slide in human studies, such as chromosome-wide aneuploidy study (CWAS)8. The basis of the method, marketed by Cytocell as the Chromoprobe Multiprobe System, is an OctoChrome device that is divided into 8 squares, each of which carries three different whole chromosome painting probes (Figure 1). Each of the three probes is directly labeled with a different colored fluorophore, green (FITC), red (Texas Red), and blue (Coumarin). The arrangement of chromosome combinations on the OctoChrome device has been designed to facilitate the identification of the non-random structural chromosome alterations (translocations) found in the most common leukemias and lymphomas, for instance t(9;22), t(15;17), t(8;21), t(14;18)9. Moreover, numerical changes (aneuploidy) in chromosomes can be detected concurrently. The corresponding template slide is also divided into 8 squares onto which metaphase spreads are bound (Figure 2), and is positioned over the OctoChrome device. The probes and target DNA are denatured at high-temperature and hybridized in a humid chamber, and then all 24 human chromosomes can be visualized simultaneously. 
OctoChrome FISH is a promising technique for the clinical diagnosis of leukemia and lymphoma and for detection of aneuploidies in all chromosomes. We have applied this new Chromosomic approach in a CWAS study of benzene-exposed Chinese workers8,10.

A novel fluorescence in situ hybridization (FISH) method that simultaneously examines both numerical and structural chromosome alterations, particularly the specific chromosomal translocations associated with leukemia and lymphoma, of all 24 human chromosomes on a single device in one hybridization, is described.

Fluorescence in situ hybridization (FISH) is a technique that allows specific DNA sequences to be detected on metaphase or interphase chromosomes in cell ...

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

Mammalian DNA replication initiates at multiple sites along chromosomes at different times during S phase, following a temporal replication program. The specification of replication timing is thought to be a dynamic process regulated by tissue-specific and developmental cues that are responsive to epigenetic modifications. However, the mechanisms regulating where and when DNA replication initiates along chromosomes remains poorly understood. Homologous chromosomes usually replicate synchronously, however there are notable exceptions to this rule. For example, in female mammalian cells one of the two X chromosomes becomes late replicating through a process known as X inactivation1. Along with this delay in replication timing, estimated to be 2-3 hr, the majority of genes become transcriptionally silenced on one X chromosome. In addition, a discrete cis-acting locus, known as the X inactivation center, regulates this X inactivation process, including the induction of delayed replication timing on the entire inactive X chromosome. In addition, certain chromosome rearrangements found in cancer cells and in cells exposed to ionizing radiation display a significant delay in replication timing of &gt;3 hours that affects the entire chromosome2,3. Recent work from our lab indicates that disruption of discrete cis-acting autosomal loci result in an extremely late replicating phenotype that affects the entire chromosome4. Additional 'chromosome engineering' studies indicate that certain chromosome rearrangements affecting many different chromosomes result in this abnormal replication-timing phenotype, suggesting that all mammalian chromosomes contain discrete cis-acting loci that control proper replication timing of individual chromosomes5.
Here, we present a method for the quantitative analysis of chromosome replication timing combined with fluorescent in situ hybridization. This method allows for a direct comparison of replication timing between homologous chromosomes within the same cell, and was adapted from6. In addition, this method allows for the unambiguous identification of chromosomal rearrangements that correlate with changes in replication timing that affect the entire chromosome. This method has advantages over recently developed high throughput micro-array or sequencing protocols that cannot distinguish between homologous alleles present on rearranged and un-rearranged chromosomes. In addition, because the method described here evaluates single cells, it can detect changes in chromosome replication timing on chromosomal rearrangements that are present in only a fraction of the cells in a population.

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

A quantitative method for the analysis of chromosome replication timing is described. The method utilizes BrdU incorporation in combination with fluorescent in situ hybridization (FISH) to assess replication timing of mammalian chromosomes. This technique allows for the direct comparison of rearranged and un-rearranged chromosomes within the same cell.

Mammalian DNA replication initiates at multiple sites along chromosomes at different times during S phase, following a temporal replication program. The ...

Combined DNA-RNA Fluorescent In situ Hybridization (FISH) to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells

Fluorescent in situ hybridization (FISH) is a molecular technique which enables the detection of nucleic acids in cells. DNA FISH is often used in cytogenetics and cancer diagnostics, and can detect aberrations of the genome, which often has important clinical implications. RNA FISH can be used to detect RNA molecules in cells and has provided important insights in regulation of gene expression. Combining DNA and RNA FISH within the same cell is technically challenging, as conditions suitable for DNA FISH might be too harsh for fragile, single stranded RNA molecules. We here present an easily applicable protocol which enables the combined, simultaneous detection of Xist RNA and DNA encoded by the X chromosomes. This combined DNA-RNA FISH protocol can likely be applied to other systems where both RNA and DNA need to be detected.

Combined DNA-RNA Fluorescent In situ Hybridization FISH to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells

Fluorescent in situ hybridization (FISH) allows the detection of nucleic acids in their native environment within cells. We here describe a protocol for the combined, simultaneous detection of RNA and DNA by means of FISH, which can be used to study X chromosome inactivation in mouse embryonic stem cells.

Fluorescent in situ hybridization (FISH) is a molecular technique which enables the detection of nucleic acids in cells. DNA FISH is often used in ...

Quick Fluorescent In Situ Hybridization Protocol for Xist RNA Combined with Immunofluorescence of Histone Modification in X-chromosome Inactivation

Combining RNA fluorescent in situ hybridization (FISH) with immunofluorescence (immuno-FISH) creates a technique that can be employed at the single cell level to detect the spatial dynamics of RNA localization with simultaneous insight into the localization of proteins, epigenetic modifications and other details which can be highlighted by immunofluorescence. X-chromosome inactivation is a paradigm for long non-coding RNA (lncRNA)-mediated gene silencing. X-inactive specific transcript (Xist) lncRNA accumulation (called an Xist cloud) on one of the two X-chromosomes in mammalian females is a critical step to initiate X-chromosome inactivation. Xist RNA directly or indirectly interacts with various chromatin-modifying enzymes and introduces distinct epigenetic landscapes to the inactive X-chromosome (Xi). One known epigenetic hallmark of the Xi is the Histone H3 trimethyl-lysine 27 (H3K27me3) modification. Here, we describe a simple and quick immuno-FISH protocol for detecting Xist RNA using RNA FISH with multiple oligonucleotide probes coupled with immunofluorescence of H3K27me3 to examine the localization of Xist RNA and associated epigenetic modifications. Using oligonucleotide probes results in a shorter incubation time and more sensitive detection of Xist RNA compared to in vitro transcribed RNA probes (riboprobes). This protocol provides a powerful tool for understanding the dynamics of lncRNAs and its associated epigenetic modification, chromatin structure, nuclear organization and transcriptional regulation.

Quick Fluorescent In Situ Hybridization Protocol for Xist RNA Combined with Immunofluorescence of Histone Modification in X-chromosome Inactivation

We developed an easily customized strand-specific fluorescent in situ hybridization (FISH) protocol combined with immunofluorescence. This allows for a detailed examination of RNA dynamics with simultaneous insight into the chromatin structure, nuclear organization, and transcriptional regulation at the single cell level.

Combining RNA fluorescent in situ hybridization (FISH) with immunofluorescence (immuno-FISH) creates a technique that can be employed at the single cell ...

Exploring X Chromosomal Aberrations in Ovarian Cells by Using Fluorescence In Situ Hybridization

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