This work was supported by NIH Grant GM59354 awarded to Sharon E. Bickel. We thank Huy Q. Nguyen for assistance in developing the protocol for generating fluorescent arm probes, Ann Lavanway for help with confocal microscopy, and J. Emiliano Reed for technical assistance. We also thank numerous colleagues in the Drosophila community for helpful discussions and advice.

<ol>
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B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disjunction+of+homologous+chromosomes+in+meiosis+I+depends+on+proteolytic+cleavage+of+the+meiotic+cohesin+Rec8+by+separin.">Disjunction of homologous chromosomes in meiosis I depends on proteolytic cleavage of the meiotic cohesin Rec8 by separin.</a> Cell. 103 (3), 387-398 (2000).</li><li>Bickel, S. E., Orr-Weaver, T., Balicky, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+sister-chromatid+cohesion+protein+ORD+is+required+for+chiasma+maintenance+in+Drosophila+oocytes.">The sister-chromatid cohesion protein ORD is required for chiasma maintenance in Drosophila oocytes.</a> Curr. Biol. 12 (11), 925-929 (2002).</li><li>Hodges, C. A., Revenkova, E., Jessberger, R., Hassold, T. J., Hunt, P. 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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defective+cohesin+is+associated+with+age-dependent+misaligned+chromosomes+in+oocytes.">Defective cohesin is associated with age-dependent misaligned chromosomes in oocytes.</a> Reprod Biomed Online. 16 (1), 103-112 (2008).</li><li>Subramanian, V. V., Bickel, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aging+predisposes+oocytes+to+meiotic+nondisjunction+when+the+cohesin+subunit+SMC1+is+reduced.">Aging predisposes oocytes to meiotic nondisjunction when the cohesin subunit SMC1 is reduced.</a> PLoS Genet. 4 (11), e1000263 (2008).</li><li>Chiang, T., Duncan, F. E., Schindler, K., Schultz, R. M., Lampson, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+that+weakened+centromere+cohesion+is+a+leading+cause+of+age-related+aneuploidy+in+oocytes.">Evidence that weakened centromere cohesion is a leading cause of age-related aneuploidy in oocytes.</a> Curr. Biol. 20 (17), 1522-1528 (2010).</li><li>Lister, L. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-related+meiotic+segregation+errors+in+mammalian+oocytes+are+preceded+by+depletion+of+cohesin+and+Sgo2.">Age-related meiotic segregation errors in mammalian oocytes are preceded by depletion of cohesin and Sgo2.</a> Curr. Biol. 20 (17), 1511-1521 (2010).</li><li>Duncan, F. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosome+cohesion+decreases+in+human+eggs+with+advanced+maternal+age.">Chromosome cohesion decreases in human eggs with advanced maternal age.</a> Aging Cell. 11 (6), 1121-1124 (2012).</li><li>Yun, Y., Lane, S. I., Jones, K. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Premature+dyad+separation+in+meiosis+II+is+the+major+segregation+error+with+maternal+age+in+mouse+oocytes.">Premature dyad separation in meiosis II is the major segregation error with maternal age in mouse oocytes.</a> Development. 141 (1), 199-208 (2014).</li><li>Subramanian, V. V., Bickel, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterochromatin-mediated+association+of+achiasmate+homologues+declines+with+age+when+cohesion+is+compromised.">Heterochromatin-mediated association of achiasmate homologues declines with age when cohesion is compromised.</a> Genetics. 181, 1207-1218 (2009).</li><li>Jeffreys, C. A., Burrage, P. S., Bickel, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+model+system+for+increased+meiotic+nondisjunction+in+older+oocytes.">A model system for increased meiotic nondisjunction in older oocytes.</a> Curr. Biol. 13 (6), 498-503 (2003).</li><li>Weng, K. A., Jeffreys, C. A., Bickel, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rejuvenation+of+Meiotic+Cohesion+in+Oocytes+during+Prophase+I+Is+Required+for+Chiasma+Maintenance+and+Accurate+Chromosome+Segregation.">Rejuvenation of Meiotic Cohesion in Oocytes during Prophase I Is Required for Chiasma Maintenance and Accurate Chromosome Segregation.</a> PLoS Genetics. 10 (9), e1004607 (2014).</li><li>Perkins, A. T., Das, T. M., Panzera, L. C., Bickel, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidative+stress+in+oocytes+during+midprophase+induces+premature+loss+of+cohesion+and+chromosome+segregation+errors.">Oxidative stress in oocytes during midprophase induces premature loss of cohesion and chromosome segregation errors.</a> Proc Natl Acad Sci U S A. 113 (44), E6823-E6830 (2016).</li><li>Dernburg, A. F., Sedat, J. W., Hawley, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+evidence+of+a+role+for+heterochromatin+in+meiotic+chromosome+segregation.">Direct evidence of a role for heterochromatin in meiotic chromosome segregation.</a> Cell. 86, 135-146 (1996).</li><li>Dernburg, A. F., Sedat, J. 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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterochromatin-Associated+Proteins+HP1a+and+Piwi+Collaborate+to+Maintain+the+Association+of+Achiasmate+Homologs+in+Drosophila+Oocytes.">Heterochromatin-Associated Proteins HP1a and Piwi Collaborate to Maintain the Association of Achiasmate Homologs in Drosophila Oocytes.</a> Genetics. 203 (1), 173-189 (2016).</li><li>Joyce, E. F., Apostolopoulos, N., Beliveau, B. J., Wu, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Germline+progenitors+escape+the+widespread+phenomenon+of+homolog+pairing+during+Drosophila+development.">Germline progenitors escape the widespread phenomenon of homolog pairing during Drosophila development.</a> PLoS Genet. 9 (12), e1004013 (2013).</li><li>Gilliland, W. D., Hughes, S. F., Vietti, D. R., Hawley, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Congression+of+achiasmate+chromosomes+to+the+metaphase+plate+in+Drosophila+melanogaster+oocytes.">Congression of achiasmate chromosomes to the metaphase plate in Drosophila melanogaster oocytes.</a> Dev Biol. 325 (1), 122-128 (2009).</li><li>Gyuricza, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+and+Stable+Cohesins+Regulate+Synaptonemal+Complex+Assembly+and+Chromosome+Segregation.">Dynamic and Stable Cohesins Regulate Synaptonemal Complex Assembly and Chromosome Segregation.</a> Curr Biol. 26 (13), 1688-1698 (2016).</li><li>Radford, S. J., McKim, K. 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The authors declare no competing financial interests.

The use of FISH probes to assess the state of arm cohesion in prometaphase I and metaphase I Drosophila oocytes is a significant advancement in the field of Drosophila meiosis. Historically, Drosophila researchers have been limited to genetic tests to infer premature loss of arm cohesion in mature oocytes11,18,19. Now, with the methods presented here, the state of arm cohesion can be assayed directly using FISH. The ability to obtain physical evidence for premature loss of arm cohesion greatly expands the repertoire of approaches available to study the mechanisms that lead to premature loss of arm cohesion and chromosome missegregation in Drosophila oocytes.
Critical Steps 
Although we have not performed a systematic analysis of critical parameters necessary for successful visualization of a fluorescent probe that hybridizes to single copy sequences along the chromosome arm in mature Drosophila oocytes, we offer the following list of factors that may have contributed to the success of this technique. To generate the arm probe, we used a combination of six overlapping BAC probes that cover an interval of approximately 0.8 Mb on the X-chromosome arm. Our initial attempts using fewer BACs that spanned a smaller region were not successful. In addition, we have used a method to fragment and amplify the BAC DNA before end-labeling with Alexa-647. The majority of restriction fragments that were labeled were 100 - 200 bp long (Figure 2), which agrees well with the target size of 150 nucleotides that is necessary for efficient diffusion through thick tissues21. Fixation conditions that preserve morphology of the large Drosophila oocyte but also allow probe penetration are essential. We have modified our previously used fixation method23 by adding room temperature heptane to a fix solution preheated to 37 &#176;C. We think it possible that both the addition of heptane to increase penetration through the vitelline membrane as well as the lowered temperature for fixation contributed to successful fixation conditions for visualization of arm cohesion.
When assaying for premature loss of cohesion, one requires a sample size that allows quantification of defects that are present at low to moderate levels. To obtain large numbers of mature oocytes, others have used blender disruption of adult females combined with sieving 26,27. Here we describe a method to hand dissect ovaries from 20 - 25 females, with manual separation of late stage oocytes by pipetting. While the blender/sieving method should also work with this protocol, one advantage of hand dissection is that without the sieving step, oocytes spend less time in buffer before fixation, which decreases the chance of anaphase I onset due to artificial egg activation. In addition, this method requires fewer females as starting material, uses smaller volumes of reagents, and has a simpler workflow, all of which facilitate processing multiple genotypes.
Hand dissection and manual separation of mature oocytes provides sufficient quantities of oocytes to &#34;roll&#34; for chorion and vitelline membrane removal and results in approximately 75 - 150 oocytes at the end of the hybridization washes. One trick to maximize the yield of metaphase I arrested oocytes is to hold virgin females in yeasted vials in the absence of males before dissection25. Oocyte rolling to remove chorions and vitelline membranes must be performed with a gentle touch in order to avoid destroying oocytes. However, removing chorions and vitelline membranes from 100% of the oocytes is not a realistic goal and excessive rolling only results in loss of oocytes. Therefore, each rolling cycle should be stopped when approximately 75 - 85% of oocytes lack chorions and vitelline membranes.
Limitations 
Despite our success at generating and using arm probes to monitor the state of cohesion in mature Drosophila oocytes, the procedure still suffers from limitations. While we rarely failed to detect a signal for the pericentromeric probe, the arm probe signal was weak or absent in approximately 15 - 20% of the oocytes that we imaged. One contributing factor may be differential penetration of the pericentric oligonucleotide probe (22 - 28 nucleotides) versus the larger arm probe (100 - 200 nucleotides). In addition, the large repetitive sequence recognized by the pericentric probe results in a signal that is brighter than that for the arm probe. The orientation of the oocyte and placement of meiotic chromosomes within the oocyte also present a challenge when imaging. Although the meiotic chromosomes are relatively close to the cell cortex, it is impossible to control the orientation of the oocyte when mounting on the coverslip. Therefore, in some fraction of the oocytes, the meiotic chromosomes may be 100 - 200 &#181;m from the coverslip and the signal intensity and quality may be negatively impacted when trying to image through the bulk of the oocyte. These limitations mean that the number of oocytes included in the final quantitative analysis is considerably lower than the number of oocytes processed in the protocol. Determining the state of arm cohesion in 100 oocytes will most likely require two independent preparations. An additional limiting factor in quantifying cohesion defects using a laser scanning confocal microscope is the time required to capture a typical Z series (approximately 5 min), even when the captured area is confined to 1,024 x 512 pixels. This means that comparison of cohesion in control and experimental genotypes (100 oocytes each) will necessitate approximately 16 h of image collection time.
Modifications and Troubleshooting 
We have been able to monitor the state of arm cohesion using a probe that recognizes the X chromosome at cytological position 6E-7B19. A probe that hybridizes to a more distal location on the X chromosome may be more desirable for detecting the failure of chiasma maintenance. In addition, other researchers may wish to analyze arm cohesion on other chromosomes. While we have not attempted to probe the autosomes, the preliminary data indicate that a probe that recognizes the 2F-3C region of the X chromosome also results in a detectable signal. However, based on limited preliminary data, the signal of the 2F-3C probe may be less &#34;compact&#34; than that for the 6E-7B probe. Although the FISH signals are tight and crisp for some oocyte chromosomes, the hybridization signal on the chromosomes of other oocytes is considerably more diffuse. Whether these differences in signal reflect genuine differences in chromosome morphology between the two cytological locations, variability in the hybridization efficiency of the two probes, or just stochastic variability between different oocytes will require a more thorough analysis.
Importantly, even for the 6E-7B probe, we observed a diffuse chromosomal signal in some oocytes, and this often necessitated their exclusion from analysis. In addition, we have noticed variation in signal quality and brightness between different preparations of the 6E-7B probe and this required that imaging parameters be adjusted accordingly. Raising the hybridization temperature to 42 &#176;C did not reduce the number of oocytes with diffuse signal, but it did severely impact the signal for the pericentric oligonucleotide probe. In an effort to better preserve chromosome morphology24, we also tried a longer denaturation step at a lower temperature (80 &#176;C), but found that this treatment neither increased the signal intensity nor reduced the number of oocytes with diffuse chromosomal FISH signal. We also attempted to obtain a brighter arm signal by using 12 overlapping BACs corresponding to an approximately 1.5 Mb interval that spanned cytological region 5E-7B. When using a 12 BAC probe, the degree of variation in signal intensity as well as the percentage of oocytes lacking signal was no different than when six BACs were used to make the arm probe. It was also more challenging to score for cohesion defects when using the 12 BAC probe because the FISH signals were larger, making it more difficult to resolve spots corresponding to individual chromatids. In cases in which no signal is obtained with a newly prepared probe, researchers should check the size of the amplified and restriction digested BAC DNA to confirm that the fragments used for end labeling are primarily within the 100 - 200 bp range. This is likely to be one of the most critical factors in successful probe preparation.
Future Directions 
Future work to enhance image acquisition as well as adapting the protocol to include immunostaining would be major improvements that would benefit other researchers. During image collection, collecting a greater number of images in the axial dimension as well as faster image acquisition would be advantageous for quantifying cohesion defects. In optimizing the imaging parameters for the laser scanning confocal, we found that 0.25 &#181;m was the smallest step size that could be used for the Z series in order to avoid appreciable bleaching of the FISH signals. However, for FISH signals that are separated by less than 0.25 &#181;m in the Z stack, this step size may result in failure to capture the &#34;space&#34; between the foci and may therefore underestimate the number of cohesion defects. The faster image acquisition speed of a spinning disc confocal may allow smaller step sizes to be used without signal bleaching. This would provide more accurate image reconstruction in the axial dimension as well as permit larger number of oocytes to be analyzed for cohesion defects. In addition, the ability to combine immunostaining with FISH visualization of the state of arm cohesion would significantly enhance the protocol. For a number of preparations, we tried to perform spindle immunostaining following the hybridization procedure. However, robust spindle staining was visible in only a small fraction of the oocytes. Moreover, for reasons that are not clear, higher levels of spurious FISH signals surrounded the meiotic chromosomes when FISH and immunostaining were combined. Further work to optimize the protocol to allow immunostaining either before or after the FISH procedure would greatly expand the capability of this technique.

Proper segregation of chromosomes during mitosis and meiosis requires that sister chromatid cohesion be established, maintained, and released in a coordinated fashion1,2. Cohesion is established during S phase and is mediated by the cohesin complex, which forms physical linkages that hold the sister chromatids together. In meiosis, cohesion distal to a crossover also functions to hold recombinant homologs together and this physical association helps ensure proper orientation of the bivalent on the metaphase I spindle (Figure 1)3,4,5. Release of arm cohesion at anaphase I allows the homologs to segregate to opposite spindle poles. However, if arm cohesion is lost prematurely, recombinant homologs will lose their physical connection and segregate randomly, which can result in aneuploid gametes (Figure 1).
In human oocytes, errors in chromosome segregation are the leading cause of miscarriages and birth defects, such as Down Syndrome6, and their incidence increases exponentially with maternal age7. Sister chromatid cohesion is established in fetal oocytes and meiotic recombination is completed before birth. Oocytes then arrest in mid-prophase I until ovulation and during this arrest, the continued physical association of recombinant homologs relies on sister chromatid cohesion. Therefore, accurate segregation during meiosis and normal pregnancy outcomes require that cohesion remain intact for up to five decades.
Premature loss of cohesion during the prolonged meiotic arrest of human oocytes has been proposed to contribute to the maternal age effect and multiple lines of evidence support this hypothesis8,9. However, given the challenges of studying meiotic cohesion in human oocytes, much of our understanding of this phenomenon relies on the use of model organisms5,10,11,12,13,14,15.
Drosophila melanogaster oocytes offer numerous advantages for the study of meiotic cohesion and chromosome segregation. A simple genetic assay allows one to recover progeny from aneuploid gametes and measure the fidelity of X-chromosome segregation on a large scale11,16,17. Moreover, one may also determine whether chromosome segregation errors arise because recombinant homologs missegregate during meiosis I, a phenotype that is consistent with premature loss of arm cohesion11,18,19. Direct observation of the state of meiotic cohesion in Drosophila oocytes is also possible using fluorescence in situ hybridization (FISH). Although fluorescent oligonucleotides that hybridize to repetitive satellite sequences have been used for over a decade to monitor pericentromeric cohesion in mature Drosophila oocytes4,20, analysis of arm cohesion has been much more challenging. Visualization of the state of arm cohesion requires a probe that spans a large region of single copy sequences and is bright enough to result in visible signals for individual sister chromatids when arm cohesion is absent. In addition, the oocyte fixation conditions and size of the labeled DNAs must facilitate penetration21 into the large mature Drosophila oocyte (200 &#181;m wide by 500 &#181;m long). Recently, an arm probe was successfully utilized to visualize Drosophila oocyte chromatids during anaphase I, but the authors stated that they could not detect a signal in prometaphase or metaphase I arrested oocytes22. Here we provide a detailed protocol for the generation of X-chromosome arm FISH probes and oocyte preparation conditions that have allowed us to assay for premature loss of sister chromatid cohesion in prometaphase I and metaphase I oocytes. These techniques, which have enabled us to identify gene products that are required for the maintenance of meiotic cohesion, will allow others to assay for sister chromatid cohesion defects in mature Drosophila oocytes of different genotypes.

<table>
	<tbody>
		<tr>
			<td>Kits</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Midi Prep kit</td>
			<td>Qiagen</td>
			<td>12143</td>
			<td>Prep BAC clone DNA</td>
		</tr>
		<tr>
			<td>GenomePlex Complete Whole Genome Amplification (WGA) Kit</td>
			<td>Sigma</td>
			<td>WGA2</td>
			<td>Amplify BAC clone DNA</td>
		</tr>
		<tr>
			<td>ARES Alexa Fluor 647 DNA labeling kit</td>
			<td>Invitrogen</td>
			<td>A21676</td>
			<td>Label BAC clone DNA</td>
		</tr>
		<tr>
			<td>PCR purification kit</td>
			<td>Qiagen</td>
			<td>28104</td>
			<td>Remove non-conjugated dye following labeling of BAC clone DNA</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Chemicals &#38; Solutions</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Note: All solutions are prepared using sterile ultrapure water and should be sterilized either by autoclave or filter sterilization.</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Fisher Scientific</td>
			<td>BP1600-100</td>
			<td>Prepare 10% stock 
			Freeze aliquots</td>
		</tr>
		<tr>
			<td>Calcium chloride</td>
			<td>Fisher Scientific</td>
			<td>C75-500</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI (4&#8217;, 6-Diamidino-2-Phenylindole, Dihydrochloride)</td>
			<td>Invitrogen</td>
			<td>D1306</td>
			<td>Toxic: wear appropriate protection. Prepare 100&#181;g/ml stock in 100% ethanol, store in aliquots at -20 &#176;C. Prepare 1 &#181;g/ml solution in 2X SSCT before use.</td>
		</tr>
		<tr>
			<td>Dextran sulfate</td>
			<td>Sigma</td>
			<td>D-8906</td>
			<td></td>
		</tr>
		<tr>
			<td>Drierite</td>
			<td>Drierite Company</td>
			<td>23001</td>
			<td></td>
		</tr>
		<tr>
			<td>Dithiothreitol (DTT)</td>
			<td>Invitrogen</td>
			<td>15508-013</td>
			<td>Prepare 10 mM stock</td>
		</tr>
		<tr>
			<td>dTTP (10 &#181;mol, 100 &#181;l)</td>
			<td>Boehringer Mannheim</td>
			<td>1277049</td>
			<td>Prepare 1 mM stock</td>
		</tr>
		<tr>
			<td>EDTA (Disodium ethylenediamine tetraacetic acid)</td>
			<td>Fisher Scientific</td>
			<td>S311-500</td>
			<td>Prepare 250 mM stock</td>
		</tr>
		<tr>
			<td>100% ethanol (molecular grade, 200 proof)</td>
			<td>Decon Laboratories</td>
			<td>2716</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris (Ultra Pure)</td>
			<td>Invitrogen</td>
			<td>15504-020</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA (Ethylenebis(oxyethylenenitrilo)tetraacetic acid)</td>
			<td>Sigma</td>
			<td>E-3889</td>
			<td></td>
		</tr>
		<tr>
			<td>16% formaldehyde</td>
			<td>Ted Pella, Inc.</td>
			<td>18505</td>
			<td>Toxic: wear appropriate protection</td>
		</tr>
		<tr>
			<td>Formamide</td>
			<td>Invitrogen</td>
			<td>AM9342</td>
			<td>Toxic: wear appropriate protection</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Fisher Scientific</td>
			<td>D16-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycogen</td>
			<td>Roche</td>
			<td>901393</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Boehringer Mannheim</td>
			<td>737-151</td>
			<td></td>
		</tr>
		<tr>
			<td>Heptane</td>
			<td>Fisher Scientific</td>
			<td>H350-4</td>
			<td>Toxic: wear appropriate protection.</td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>Millipore</td>
			<td>HX0603-4</td>
			<td>Toxic: wear appropriate protection.</td>
		</tr>
		<tr>
			<td>Hydroxylamine</td>
			<td>Sigma</td>
			<td>438227</td>
			<td>Prepare 3 M stock</td>
		</tr>
		<tr>
			<td>4.9 M Magnesium chloride</td>
			<td>Sigma</td>
			<td>104-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4 &#376; 7H2O</td>
			<td>Fisher Scientific</td>
			<td>S373-500</td>
			<td></td>
		</tr>
		<tr>
			<td>NaH2PO4 &#376; 2H2O</td>
			<td>Fisher Scientific</td>
			<td>S369-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-lysine (0.1mg/ml)</td>
			<td>Sigma</td>
			<td>P8920-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium acetate</td>
			<td>Fisher Scientific</td>
			<td>BP364-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium acetate</td>
			<td>Fisher Scientific</td>
			<td>S209-500</td>
			<td>Prepare 3M stock</td>
		</tr>
		<tr>
			<td>Sodium cacodylate</td>
			<td>Polysciences, Inc.</td>
			<td>1131</td>
			<td>Toxic: wear appropriate protection. Prepare 400mM stock</td>
		</tr>
		<tr>
			<td>Sodium citrate</td>
			<td>Fisher Scientific</td>
			<td>BP327-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Fisher Scientific</td>
			<td>S271-3</td>
			<td>Sodium chloride</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Fisher Scientific</td>
			<td>S5-500</td>
			<td></td>
		</tr>
		<tr>
			<td>10% Tween 20</td>
			<td>Thermo Scientific</td>
			<td>28320</td>
			<td>Surfact-Amps</td>
		</tr>
		<tr>
			<td>10% Triton X-100</td>
			<td>Thermo Scientific</td>
			<td>28314</td>
			<td>Surfact-Amps</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Solutions</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Note: All solutions are prepared using sterile ultrapure water and should be sterilized either by autoclave or filter sterilization.</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TE buffer</td>
			<td></td>
			<td></td>
			<td>10 mM Tris, 1 mM EDTA, pH = 8.0</td>
		</tr>
		<tr>
			<td>20X SSC (Saline Sodium Citrate)</td>
			<td></td>
			<td></td>
			<td>3 M NaCl, 300 mM sodium citrate</td>
		</tr>
		<tr>
			<td>2X cacodylate fix solution</td>
			<td></td>
			<td></td>
			<td>Toxic: wear appropriate protection. 200 mM sodium cacodylate, 200 mM sucrose, 80 mM sodium acetate, 20 mM EGTA</td>
		</tr>
		<tr>
			<td>1.1X Hybridization buffer</td>
			<td></td>
			<td></td>
			<td>3.3X SSC, 55% formamide, 11% dextran sulfate</td>
		</tr>
		<tr>
			<td>Fix solution</td>
			<td></td>
			<td></td>
			<td>Toxic: wear appropriate protection. 4% formaldehyde, 1X cacodylate fix solution</td>
		</tr>
		<tr>
			<td>PBSBTx</td>
			<td></td>
			<td></td>
			<td>1X PBS, 0.5% BSA, 0.1% Trition X-100</td>
		</tr>
		<tr>
			<td>PBSTx</td>
			<td></td>
			<td></td>
			<td>1X PBS, 1% Trition X-100</td>
		</tr>
		<tr>
			<td>Extraction buffer (PBSTx + Rnase)</td>
			<td></td>
			<td></td>
			<td>1X PBS, 1% Trition X-100, 100 &#181;g/mL RNase</td>
		</tr>
		<tr>
			<td>2X SSCT</td>
			<td></td>
			<td></td>
			<td>2X SSC, 0.1% Tween 20</td>
		</tr>
		<tr>
			<td>2X SSCT + 20% formamide</td>
			<td></td>
			<td></td>
			<td>Toxic: wear appropriate protection. 2X SSC, 0.1% Tween 20, 20% formamide</td>
		</tr>
		<tr>
			<td>2X SSCT + 40% formamide</td>
			<td></td>
			<td></td>
			<td>Toxic: wear appropriate protection. 2X SSC, 0.1% Tween 20, 40% formamide</td>
		</tr>
		<tr>
			<td>2X SSCT + 50% formamide</td>
			<td></td>
			<td></td>
			<td>Toxic: wear appropriate protection. 2X SSC, 0.1% Tween 20, 50% formamide</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Enzymes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>AluI</td>
			<td>New England Biolabs</td>
			<td>R0137S</td>
			<td></td>
		</tr>
		<tr>
			<td>HaeIII</td>
			<td>New England Biolabs</td>
			<td>R0108S</td>
			<td></td>
		</tr>
		<tr>
			<td>MseI</td>
			<td>New England Biolabs</td>
			<td>R0525S</td>
			<td></td>
		</tr>
		<tr>
			<td>MspI</td>
			<td>New England Biolabs</td>
			<td>R0106S</td>
			<td></td>
		</tr>
		<tr>
			<td>RsaI</td>
			<td>New England Biolabs</td>
			<td>R0167S</td>
			<td></td>
		</tr>
		<tr>
			<td>BfuCI</td>
			<td>New England Biolabs</td>
			<td>R0636S</td>
			<td></td>
		</tr>
		<tr>
			<td>100X BSA</td>
			<td>New England Biolabs</td>
			<td></td>
			<td>Comes with NEB enzymes</td>
		</tr>
		<tr>
			<td>10X NEB buffer #2</td>
			<td>New England Biolabs</td>
			<td></td>
			<td>Restriction enzyme digestion buffer. Comes with NEB enzymes</td>
		</tr>
		<tr>
			<td>Terminal deoxynucleotidyl transferase (TdT) 400 U/&#181;l</td>
			<td>Roche/Sigma</td>
			<td>3333566001</td>
			<td></td>
		</tr>
		<tr>
			<td>TdT buffer</td>
			<td>Roche/Sigma</td>
			<td></td>
			<td>Comes with TdT enzyme</td>
		</tr>
		<tr>
			<td>Cobalt chloride</td>
			<td>Roche/Sigma</td>
			<td></td>
			<td>Toxic: wear appropriate protection. Comes with TdT enzyme</td>
		</tr>
		<tr>
			<td>RNase A (10 mg/mL)</td>
			<td>Thermo-Scientific</td>
			<td>EN0531</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Cytology Tools etc.</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Dumont</td>
			<td></td>
			<td>#5 INOX, Biologie</td>
		</tr>
		<tr>
			<td>9&#8221; Disposable glass Pasteur pipettes</td>
			<td>Fisher</td>
			<td>13-678-20C</td>
			<td>Autoclave to sterilize</td>
		</tr>
		<tr>
			<td>Shallow glass dissecting dish</td>
			<td></td>
			<td></td>
			<td>Custom made</td>
		</tr>
		<tr>
			<td>Deep well dish (3 wells)</td>
			<td>Pyrex</td>
			<td>7223-34</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherfinest Premium microscope slides</td>
			<td>Fisher Scientific</td>
			<td>22-038-104</td>
			<td>Used to cover deep well dishes</td>
		</tr>
		<tr>
			<td>Frosted glass slides, 25 x 75 mm</td>
			<td>VWR Scientific</td>
			<td>48312-002</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass slides, 3 x 1 in, 1 mm thick</td>
			<td>Thermo-Scientific</td>
			<td>3051</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslips, 10 x 10 mm, No. 1.5</td>
			<td>Thermo-Scientific</td>
			<td>3405</td>
			<td></td>
		</tr>
		<tr>
			<td>Tungsten needle</td>
			<td></td>
			<td></td>
			<td>homemade</td>
		</tr>
		<tr>
			<td>Prolong GOLD mounting media</td>
			<td>Molecular Probes</td>
			<td>P36930</td>
			<td></td>
		</tr>
		<tr>
			<td>Compressed-air in can</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PCR machine</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nanodrop 2000, spectrophotometer</td>
			<td>Thermo-Scientific</td>
			<td></td>
			<td>microvolume spectrophotometer</td>
		</tr>
		<tr>
			<td>Vortexer</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Table top microfuge at room temperature</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Table top microfuge at 4 &#176;C</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Heat block</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hybridization oven or incubator with rotator</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nutator</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>A1RSi laser scanning confoal</td>
			<td>Nikon</td>
			<td></td>
			<td>40X oil Plan Fluor DIC (NA 1.3)</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Consumables etc.</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL conical tubes</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL conical tubes</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL microfuge tubes</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>500 &#181;l microfuge tubes</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>200 &#181;l PCR tubes</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic container with tight fitting lid</td>
			<td>Various</td>
			<td></td>
			<td>To hold Drierite</td>
		</tr>
		<tr>
			<td>Kimwipes</td>
			<td>Various</td>
			<td></td>
			<td>disposable wipes</td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Various</td>
			<td></td>
			<td>paraffin film</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Other</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HPLC purified 5&#39;-labeled oligonucleotides</td>
			<td>Integrated DNA Technologies</td>
			<td></td>
			<td>Cy3-labeled probes that recognize the 359 bp satellite repeat of the X chromosome</td>
		</tr>
		<tr>
			<td>Volocity 3D Image Analysis Software</td>
			<td>PerkinElmer</td>
			<td></td>
			<td>Version 6.3</td>
		</tr>
	</tbody>
</table>

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.

Figure 5 presents images obtained with an arm probe that hybridizes to cytological region 6E-7B on the X chromosome. This probe results in a signal that co-localizes with that of DAPI, is easily distinguishable from the background, and has been used successfully to quantify arm cohesion defects in different genotypes19. Quantification of cohesion defects was restricted to prometaphase I and metaphase I stages; oocytes prior to nuclear envelope breakdown were excluded from analysis19. An intact nuclear envelope is evident when scanning in the DAPI channel because the oocyte chromosomes will be surrounded by a discrete circular area that is darker than the surrounding cytoplasm.
Figure 5B shows maximum intensity projections of individual Z series after deconvolution and contrast enhancement. Quantification of cohesion defects requires determining for each probe the number of FISH signals that overlap with the DAPI signal. For such an analysis, visualization of the merged images in three dimensions is imperative. Only FISH spots that are clearly associated with the DAPI signal in all three dimensions should be included in the analysis.
Intact sister chromatid cohesion is evidenced as two FISH spots for both the arm and pericentric probes (Figure 5B, left). Oocytes containing three or four separated FISH spots for either probe are considered to have cohesion defects (Figure 5B, right). To be classified as individual spots, two FISH signals must be minimally separated in all three dimensions by a distance corresponding to the diameter of the smallest spot. Thin faint threads connecting two FISH spots are not uncommon. Such signals are not considered completely separated and therefore are not counted as instances of cohesion defects.
With the 6E-7B arm hybridization probe, approximately 15 - 20% of the oocytes imaged completely lacked signal or the signal was too weak to confidently score. In addition, roughly 5% of the oocytes imaged exhibited a large area of diffuse chromosomal signal and these were discarded from the analysis. In contrast, it was rare to encounter oocytes for which the pericentric hybridization signal was missing or too diffuse to score.
The 6E-7B arm probe has been used extensively to quantify arm cohesion defects in prometaphase and metaphase I arrested Drosophila oocytes of different genotypes19. When the cohesin subunit SMC3 was knocked down during mid-prophase, 16.3% of the mature oocytes analyzed contained greater than two arm spots, indicative of premature loss of arm cohesion. In contrast, we observed separated arm spots in only 5.1% of oocytes that contained normal levels of SMC319. Interestingly, although the frequency of arm cohesion defects was elevated in multiple knockdown genotypes, sister chromatids were rarely separated in their pericentric regions19.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56802/56802fig1.jpg" /> 
Figure 1: Age-dependent loss of cohesion during the extended prophase I arrest of female meiosis can result in errors in chromosome segregation and aneuploidy in the egg. Cohesion is represented by black bars between the sister chromatids. Arm cohesion functions to hold recombinant homologs together and is required for accurate segregation at anaphase I. If arm cohesion is lost prematurely, chromosome missegregation can occur, resulting in an aneuploid egg. This figure was adapted from reference19. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56802/56802fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56802/56802fig2.jpg" /> 
Figure 2: Size of DNA molecules after BAC fragmentation and amplification (left) and after restriction enzyme digestion (right) for three different BACs. 400 ng of amplified DNA products are shown in the first three lanes. The last three lanes show DNA fragments after the overnight restriction digest. The amplified DNA products range from 200 bp up to 3 kb. After the restriction digest, most fragments ranged from 100 - 200 bp, but larger fragments (up to 500 bp) were visible. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56802/56802fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56802/56802fig3.jpg" /> 
Figure 3: Cytology tools. Tools used in protocol: (1) pair of forceps (#5 Dumont); (2) tungsten needle; (3) deep well dish with cover; (4) shallow glass dissecting dish; (5) pulled Pasteur pipette. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56802/56802fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56802/56802fig4.jpg" /> 
Figure 4. Arrangement and movement of slides for rolling oocytes. (A) Diagram of the slide set up for rolling of oocytes as described in the protocol. The darker area denotes the frosted glass part of the slide. (B) Direction of movement vectors for slide #2 during oocyte rolling. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56802/56802fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56802/56802fig5.jpg" /> 
Figure 5. FISH assay results. (A) Schematic depicts the Drosophila X chromosome and where the two arm probes (6 BACs each) and the 22 oligonucleotide pericentromeric probe described in the protocol hybridize. For simplicity, the 6E-7B probe is labeled 7B and the 2F-3C probe is labeled 3C. (B) Representative images of FISH results (6E-7B probe) for intact arm cohesion (two arm probe spots, one per homolog) and for disrupted arm cohesion (three to four arm probe spots). DNA is blue, the arm probe signal is yellow, and the pericentric probe signal is red. Images correspond to maximum intensity projections of Z series after deconvolution and contrast enhancement. Scale bar = 2 &#181;m. This figure was adapted from reference19. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56802/56802fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Using Fluorescence In Situ Hybridization FISH to Monitor the State of Arm Cohesion in Prometaphase and Metaphase I Drosophila Oocytes at JoVE.com

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

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.

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 ...

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11.3K Views.  Dartmouth College. 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.

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

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

Organogenesis is the study of how organs are specified and then acquire their specific shape and functions during development. The Xenopuslaevis embryo is very useful for studying organogenesis because their large size makes them very suitable for identifying organs at the earliest steps in organogenesis. At this time, the primary method used for identifying a specific organ or primordium is whole mount in situ hybridization with labeled antisense RNA probes specific to a gene that is expressed in the organ of interest. In addition, it is relatively easy to manipulate genes or signaling pathways in Xenopus and in situ hybridization allows one to then assay for changes in the presence or morphology of a target organ. Whole mount in situ hybridization is a multi-day protocol with many steps involved. Here we provide a simplified protocol with reduced numbers of steps and reagents used that works well for routine assays. In situ hybridization robots have greatly facilitated the process and we detail how and when we utilize that technology in the process. Once an in situ hybridization is complete, capturing the best image of the result can be frustrating. We provide advice on how to optimize imaging of in situ hybridization results. Although the protocol describes assessing organogenesis in Xenopus laevis, the same basic protocol can almost certainly be adapted to Xenopus tropicalis and other model systems.

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

The Xenopus laevis embryo continues to be exceptionally useful in the study of early development due to its large size and ease of manipulation. A simplified protocol for whole mount in situ hybridization protocol is provided that can be used in the identification of specific organs in this model system.

Organogenesis is the study of how organs are specified and then acquire their specific shape and functions during development.  The Xenopuslaevis embryo ...

Multi-target Chromogenic Whole-mount In Situ Hybridization for Comparing Gene Expression Domains in Drosophila Embryos

To analyze gene regulatory networks active during embryonic development and organogenesis it is essential to precisely define how the different genes are expressed in spatial relation to each other in situ. Multi-target chromogenic whole-mount in situ hybridization (MC-WISH) greatly facilitates the instant comparison of gene expression patterns, as it allows distinctive visualization of different mRNA species in contrasting colors in the same sample specimen. This provides the possibility to relate gene expression domains topographically to each other with high accuracy and to define unique and overlapping expression sites. In the presented protocol, we describe a MC-WISH procedure for comparing mRNA expression patterns of different genes in Drosophila embryos. Up to three RNA probes, each specific for another gene and labeled by a different hapten, are simultaneously hybridized to the embryo samples and subsequently detected by alkaline phosphatase-based colorimetric immunohistochemistry. The described procedure is detailed here for Drosophila, but works equally well with zebrafish embryos.

Multi-target Chromogenic Whole-mount In Situ Hybridization for Comparing Gene Expression Domains in Drosophila Embryos

We describe a multi-target chromogenic whole-mount in situ hybridization (MC-WISH) procedure in intact Drosophila embryos allowing simultaneous and specific detection of three different mRNA distribution patterns by contrasting color precipitates.

To analyze gene regulatory networks active during embryonic development and organogenesis it is essential to precisely define how the different genes are ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the Notch receptor with ligands from neighboring cells induces activation of the signaling pathway (trans-activation), while interaction with ligands from the same cell inhibits signaling (cis-inhibition). Proper balance between trans-activation and cis-inhibition helps establish optimal levels of Notch signaling in some contexts during animal development. Because of the overlapping expression domains of Notch and its ligands in many cell types and the existence of feedback mechanisms, studying the effects of a given post-translational modification on trans- versus cis-interactions of Notch and its ligands in vivo is difficult. Here, we describe a protocol for using Drosophila S2 cells in cell-aggregation assays to assess the effects of knocking down a Notch pathway modifier on the binding of Notch to each ligand in trans and in cis. S2 cells stably or transiently transfected with a Notch-expressing vector are mixed with cells expressing each Notch ligand (S2-Delta or S2-Serrate). Trans-binding between the receptor and ligands results in the formation of heterotypic cell aggregates and is measured in terms of the number of aggregates per mL composed of &#62;6 cells. To examine the inhibitory effect of cis-ligands, S2 cells co-expressing Notch and each ligand are mixed with S2-Delta or S2-Serrate cells and the number of aggregates is quantified as described above. The relative decrease in the number of aggregates due to the presence of cis-ligands provides a measure of cis-ligand-mediated inhibition of trans-binding. These straightforward assays can provide semi-quantitative data on the effects of genetic or pharmacological manipulations on the binding of Notch to its ligands, and can help deciphering the molecular mechanisms underlying the in vivo effects of such manipulations on Notch signaling.

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Complexity of in vivo systems makes it difficult to distinguish between the activation and inhibition of Notch receptor by trans- and cis-ligands, respectively. Here, we present a protocol based on in vitro cell-aggregation assays for qualitative and semi-quantitative evaluation of the binding of Drosophila Notch to trans-ligands vs cis-ligands.

Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the ...

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is the formation of the transient organizers, the node and notochordal plate, from cells that have passed through the primitive streak. The proper formation of these signaling centers is essential for the development of the body plan and techniques to visualize them are of high interest to mouse developmental biologists. The node and notochordal plate lie on the ventral surface of gastrulating mouse embryos around embryonic day (E) 7.5 of development. The node is a cup-shaped structure whose cells possess a single slender cilium each. The proper subcellular localization and rotation of the cilia in the node pit determines left-right asymmetry. The notochordal plate cells also possess single cilia albeit shorter than those of the node cells. The notochordal plate forms the notochord which acts as an important signaling organizer for somitogenesis and neural patterning. Because the cells of the node and notochordal plate are transiently present on the surface and possess cilia, they can be visualized using scanning electron microscopy (SEM). Among other techniques used to visualize these structures at the cellular level is whole mount immunofluorescence (WMIF) using the antibodies against the proteins that are highly expressed in the node and notochordal plate. In this report, we describe our optimized protocols to perform SEM and WMIF of the node and notochordal plate in developing mouse embryos to help in the assessment of tissue shape and cellular organization in wild-type and gastrulation mutant embryos.

The node and notochordal plate are transient signaling organizers in developing mouse embryos that can be visualized using several techniques. Here, we describe in detail how to perform two of the techniques to study their structure and morphogenesis: 1) scanning electron microscopy (SEM); and 2) whole mount immunofluorescence (WMIF).

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...

Wholemount In Situ Hybridization for Astyanax Embryos

In recent years, a draft genome for the blind Mexican cavefish (Astyanax mexicanus) has been released, revealing the sequence identities for thousands of genes. Prior research into this emerging model system capitalized on comprehensive genome-wide investigations that have identified numerous quantitative trait loci (QTL) associated with various cave-associated phenotypes. However, the ability to connect genes of interest to the heritable basis for phenotypic change remains a significant challenge. One technique that can facilitate deeper understanding of the role of development in troglomorphic evolution is whole-mount in situ hybridization. This technique can be implemented to directly compare gene expression between cave- and surface-dwelling forms, nominate candidate genes underlying established QTL, identify genes of interest from next-generation sequencing studies, or develop other discovery-based approaches. In this report, we present a simple protocol, supported by a flexible checklist, that can be widely adapted for use well beyond the presented study system. It is hoped that this protocol can serve as a broad resource for the Astyanax community and beyond.

Wholemount In Situ Hybridization for Astyanax Embryos

This protocol enables visualization of gene expression in embryonic Astyanax cavefish. This approach has been developed with the goal of maximizing gene expression signal, while minimizing non-specific background staining.

In recent years, a draft genome for the blind Mexican cavefish (Astyanax mexicanus) has been released, revealing the sequence identities for thousands of ...

Genotyping and Quantification of In Situ Hybridization Staining in Zebrafish

In situ hybridization (ISH) is an important technique that enables researchers to study mRNA distribution in situ and has been a critical technique in developmental biology for decades. Traditionally, most gene expression studies relied on visual evaluation of the ISH signal, a method that is prone to bias, particularly in cases where sample identities are known a priori. We have previously reported on a method to circumvent this bias and provide a more accurate quantification of ISH signals. Here, we present a simple guide to apply this method to quantify the expression levels of genes of interest in ISH-stained embryos and correlate that with their corresponding genotypes. The method is particularly useful to quantify spatially restricted gene expression signals in samples of mixed genotypes and it provides an unbiased and accurate alternative to the traditional visual scoring methods.

Gene editing technologies have enabled researchers to generate zebrafish mutants to investigate gene function with relative ease. Here, we provide a guide to perform parallel embryo genotyping and quantification of in situ hybridization signals in zebrafish. This unbiased approach provides greater accuracy in phenotypical analyses based on in situ hybridization.

In situ hybridization (ISH) is an important technique that enables researchers to study mRNA distribution in situ and has been a critical technique in ...

Whole-Mount In Situ Hybridization in Zebrafish Embryos and Tube Formation Assay in iPSC-ECs to Study the Role of Endoglin in Vascular Development

Vascular development is determined by the sequential expression of specific genes, which can be studied by performing in situ hybridization assays in zebrafish during different developmental stages. To investigate the role of endoglin(eng) in vessel formation during the development of hereditary hemorrhagic telangiectasia&#160;(HHT), morpholino-mediated targeted knockdown of eng in zebrafish are used to study its temporal expression and associated functions. Here, whole-mount in situ RNA hybridization (WISH) is employed for the analysis of eng and its downstream genes in zebrafish embryos. Also, tube formation assays are performed in HHT patient-derived induced pluripotent stem cell-differentiated&#160;endothelial cells (iPSC-ECs; with eng mutations). A specific signal amplifying system using the whole amount In Situ Hybridization &#8211; WISH provides higher resolution and lower background results compared to traditional methods. To obtain a better signal, the post-fixation time is adjusted to 30 min after probe hybridization. Because fluorescence staining is not sensitive in zebrafish embryos, it is replaced with diaminobezidine (DAB) staining here. In this protocol, HHT&#160;patient-derived iPSC lines containing an eng mutation are differentiated into endothelial cells. After coating a plate with basement membrane matrix for 30 min at 37 &#176;C, iPSC-ECs are seeded as a monolayer into wells and kept at 37 &#176;C for 3 h. Then, the tube length and number of branches are calculated using microscopic images. Thus, with this improved WISH protocol, it is shown that reduced eng expression affects endothelial progenitor formation in zebrafish embryos. This is further confirmed by tube formation assays using iPSC-ECs derived from a patient with HHT. These assays confirm the role for eng in early vascular development.

Presented here is a protocol for whole-mount in situ RNA hybridization analysis in zebrafish and tube formation assay in patient-derived induced pluripotent stem cell-derived endothelial cells to study the role of endoglin in vascular formation.

Vascular development is determined by the sequential expression of specific genes, which can be studied by performing in situ hybridization assays in ...

Fluorescent Calcium Imaging and Subsequent In Situ Hybridization for Neuronal Precursor Characterization in Xenopus laevis

Spontaneous intracellular calcium activity can be observed in a variety of cell types and is proposed to play critical roles in a variety of physiological processes. In particular, appropriate regulation of calcium activity patterns during embryogenesis is necessary for many aspects of vertebrate neural development, including proper neural tube closure, synaptogenesis, and neurotransmitter phenotype specification. While the observation that calcium activity patterns can differ in both frequency and amplitude suggests a compelling mechanism by which these fluxes might transmit encoded signals to downstream effectors and regulate gene expression, existing population-level approaches have lacked the precision necessary to further explore this possibility. Furthermore, these approaches limit studies of the role of cell-cell interactions by precluding the ability to assay the state of neuronal determination in the absence of cell-cell contact. Therefore, we have established an experimental workflow that pairs time-lapse calcium imaging of dissociated neuronal explants with a fluorescence in situ hybridization assay, allowing the unambiguous correlation of calcium activity pattern with molecular phenotype on a single-cell level. We were successfully able to use this approach to distinguish and characterize specific calcium activity patterns associated with differentiating neural cells and neural progenitor cells, respectively; beyond this, however, the experimental framework described in this article could be readily adapted to investigate correlations between any time-series activity profile and expression of a gene or genes of interest.

Fluorescent Calcium Imaging and Subsequent In Situ Hybridization for Neuronal Precursor Characterization in Xenopus laevis

We present a two-part protocol that combines fluorescent calcium imaging with in situ hybridization, allowing the experimenter to correlate patterns of calcium activity with gene expression profiles on a single-cell level.

Spontaneous intracellular calcium activity can be observed in a variety of cell types and is proposed to play critical roles in a variety of physiological ...

A Protocol for Immunohistochemistry and RNA In-situ Distribution within Early Drosophila Embryo

Calcium induced calcium release signaling (CICR) plays a critical role in many biological processes. Every cellular activity from cell proliferation and apoptosis, development and ageing, to neuronal synaptic plasticity and regeneration have been associated with Ryanodine receptors (RyRs). Despite the importance of calcium signaling, the exact mechanism of its function in early development is unclear. As an organism with a short gestational period, the embryos of Drosophila melanogaster are prime study subjects for investigating the distribution and localization of CICR associated proteins and their regulators during development. However, because of their lipid-rich embryos and chitin-rich chorion, their utility is limited by the difficulty of mounting embryos on glass surfaces. In this work, we introduce a practical protocol that significantly enhances the attachment of Drosophila embryo onto slides and detail methods for successful histochemistry, immunohistochemistry, and in-situ hybridization. The chrome alum gelatin slide-coating method and embryo pre-embedding method dramatically increases the yield in studying Drosophila embryo protein and RNA expression. To demonstrate this approach, we studied DmFKBP12/Calstabin, a well-known regulator of RyR during early embryonic development of Drosophila melanogaster. We identified DmFKBP12 in as early as the syncytial blastoderm stage and report the dynamic expression pattern of DmFKBP12 during development: initially as an evenly distributed protein in the syncytial blastoderm, then preliminarily localizing to the basement layer of the cortex during cellular blastoderm, before distributing in the primitive neuronal and digestion architecture during the three-gem layer phase in early gastrulation. This distribution may explain the critical role RyR plays in the vital organ systems that originate in from these layers: the suboesophageal and supraesophageal ganglion, ventral nervous system, and musculoskeletal system.

A Protocol for Immunohistochemistry and RNA In-situ Distribution within Early Drosophila Embryo

Here, we describe a protocol for detection and localization of Drosophila embryo protein and RNA from collection to pre-embedding and embedding, immunostaining, and mRNA in situ hybridization.

Calcium induced calcium release signaling (CICR) plays a critical role in many biological processes. Every cellular activity from cell proliferation and ...