Name: immunofluorescent staining for visualization of heterochromatin associated proteins in drosophila salivary glands
Uploaded: 2021-08-21T14:30:00
Description: Watch this Scientific Journal Video about Immunofluorescent Staining for Visualization of Heterochromatin Associated Proteins in Drosophila Salivary Glands at JoVE.com

We thank Marco Antonio Rosales Vega and Abel Segura for taking some of the confocal images, Carmen Mu&#241;oz for media preparation and Dr. Arturo Pimentel, M.C. Andr&#233;s Saralegui, and Dr. Chris Wood from the LMNA for advice on the use of the microscopes.
FUNDING: 
This work was supported by the Consejo Nacional de Ciencia y Tecnolog&#237;a (CONACyT) (A1-S-8239 to VV-G) and Programa de Apoyo a Proyectos de Investigaci&#243;n e Innovaci&#243;n Tecnol&#243;gica (204915 and 200118 to VV-G)

1. Third instar larvae culture
<ol>
	<li>Prepare 1 liter of standard media by adding 100 g of yeast, 100 g of unrefined whole cane sugar, 16 g of agar, 10 mL of propionic acid and 14 g of gelatin. Dissolve all ingredients except the yeast in 800 mL of tap water and then dissolve the yeast. Autoclave immediately for 30 minutes.
	<ol>
		<li>Afterward, let the media cool down to 60 &#176;C and add propionic acid to a final concentration 0.01%. Let the bottle stand until the gelatin is formed.</li>
	</ol>
	</li>
	<li>To op...

<ol>
	<li>Berger, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emil+Heitz,+a+true+epigenetics+pioneer.">Emil Heitz, a true epigenetics pioneer.</a> Nature Reviews Molecular Cell Biology. 20 (10), 572 (2019).</li><li>Irick, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+function+for+heterochromatin.">A new function for heterochromatin.</a> Chromosoma. 103 (1), 1-3 (1994).</li><li>Kasinathan, 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=Innovation+of+heterochromatin+functions+drives+rapid+evolution+of+essential+ZAD-ZNF+genes+in+Drosophila.">Innovation of heterochromatin functions drives rapid evolution of essential ZAD-ZNF genes in Drosophila.</a> Elife. , 1-31 (2020).</li><li>Lifschytz, E., Hareven, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterochromatin+markers:+Arrangement+of+obligatory+heterochromatin,+histone+genes+and+multisite+gene+families+in+the+interphase+nucleus+of+D.+melanogaster.">Heterochromatin markers: Arrangement of obligatory heterochromatin, histone genes and multisite gene families in the interphase nucleus of D. melanogaster.</a> Chromosoma. 86 (4), 443-455 (1982).</li><li>Eissenberg, J. C., Elgin, S. C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HP1a:+A+structural+chromosomal+protein+regulating+transcription.">HP1a: A structural chromosomal protein regulating transcription.</a> Trends in Genetics. 30 (3), 103-110 (2014).</li><li>Lee, Y. C. G., 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=Pericentromeric+heterochromatin+is+hierarchically+organized+and+spatially+contacts+H3K9me2+islands+in+euchromatin.">Pericentromeric heterochromatin is hierarchically organized and spatially contacts H3K9me2 islands in euchromatin.</a> PLoS Genetics. 16 (3), 1-27 (2020).</li><li>Koryakov, D. 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P., Bownes, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Staging+the+metamorphosis+of+Drosophila+melanogaster.">Staging the metamorphosis of Drosophila melanogaster.</a> Journal of Embryology and Experimental Morphology. 66 (1967), 57-80 (1981).</li><li>Larson, A. G., 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=Liquid+droplet+formation+by+HP1&#945;+suggests+a+role+for+phase+separation+in+heterochromatin.">Liquid droplet formation by HP1&#945; suggests a role for phase separation in heterochromatin.</a> Nature. 547 (7662), 236-240 (2017).</li><li>Larson, A. G., Narlikar, G. 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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterochromatin+protein+1+is+required+for+the+normal+expression+of+two+heterochromatin+genes+in+Drosophila.">Heterochromatin protein 1 is required for the normal expression of two heterochromatin genes in Drosophila.</a> Genetics. 155 (2), 699-708 (2000).</li><li>Marsano, R. M., Giordano, E., Messina, G., Dimitri, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+New+Portrait+of+Constitutive+Heterochromatin:+Lessons+from+Drosophila+melanogaster.">A New Portrait of Constitutive Heterochromatin: Lessons from Drosophila melanogaster.</a> Trends in Genetics. , (2019).</li><li>Strom, A. 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=Phase+separation+drives+heterochromatin+domain+formation.">Phase separation drives heterochromatin domain formation.</a> Nature. 547 (7662), 241-245 (2017).</li><li>Sanulli, S., 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=HP1+reshapes+nucleosome+core+to+promote+phase+separation+of+heterochromatin.">HP1 reshapes nucleosome core to promote phase separation of heterochromatin.</a> Nature. 575 (7782), 390-394 (2019).</li><li>Dialynas, G., Delabaere, L., Chiolo, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arp2/3+and+Unc45+maintain+heterochromatin+stability+in+Drosophila+polytene+chromosomes.">Arp2/3 and Unc45 maintain heterochromatin stability in Drosophila polytene chromosomes.</a> Experimental Biology and Medicine. 244 (15), 1362-1371 (2019).</li><li>Kolesnikova, T. D., 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=Induced+decondensation+of+heterochromatin+in+drosophila+melanogaster+polytene+chromosomes+under+condition+of+ectopic+expression+of+the+supressor+of+underreplication+gene.">Induced decondensation of heterochromatin in drosophila melanogaster polytene chromosomes under condition of ectopic expression of the supressor of underreplication gene.</a> Fly. 5 (3), 181-190 (2011).</li><li>Bettinger, J. C., Lee, K., Rougvie, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stage-specific+accumulation+of+the+terminal+differentiation+factor+LIN-29+during+Caenorhabditis+elegans+development.">Stage-specific accumulation of the terminal differentiation factor LIN-29 during Caenorhabditis elegans development.</a> Development. 122 (8), 2517-2527 (1996).</li><li>Messina, G., 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=Yeti,+an+essential+Drosophila+melanogaster+gene,+encodes+a+protein+required+for+chromatin+organization.">Yeti, an essential Drosophila melanogaster gene, encodes a protein required for chromatin organization.</a> Journal of Cell Science. 127 (11), 2577-2588 (2014).</li><li>Aguilar-Fuentes, J., 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=p8/TTDA+overexpression+enhances+UV-irradiation+resistance+and+suppresses+TFIIH+mutations+in+a+Drosophila+trichothiodystrophy+model.">p8/TTDA overexpression enhances UV-irradiation resistance and suppresses TFIIH mutations in a Drosophila trichothiodystrophy model.</a> PLOS Genetics. 4 (11), 1-9 (2008).</li><li>Reynaud, E., Lomeli, H., Vazquez, M., Zurita, 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+Drosophila+melanogaster+Homologue+of+the+Xeroderma+Pigmentosum+D+Gene+Product+Is+Located+in+Euchromatic+Regions+and+Has+a+Dynamic+Response+to+UV+Light-induced+Lesions+in+Polytene+Chromosomes.">The Drosophila melanogaster Homologue of the Xeroderma Pigmentosum D Gene Product Is Located in Euchromatic Regions and Has a Dynamic Response to UV Light-induced Lesions in Polytene Chromosomes.</a> Molecular Biology of the Cell. 10 (4), 1191-1203 (1999).</li><li>Farka&#353;, R., Mechler, B. 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The cellular function of eukaryotic organisms can define the 3D structure within the nucleus, which is supported by interactions between different proteins with chromatin and various molecules including RNA. In the last three years, the biological condensates that have had relevance, including heterochromatin, have taken a fundamental role in the determination of the phase separation promoting the distinct nuclear spatial organization of active and repressive chromatin 16,<sup class="xr...

Since the early studies of Emil Heitz1, heterochromatin has been considered an important regulator of cellular processes such as gene expression, meiotic and mitotic separation of chromosomes, and the maintenance of genome stability2,3,4.
Heterochromatin is mainly divided into two types: constitutive heterochromatin that characteristically defines repetitive sequences, and transposable elements that are present at specific chromosome sites such as the telomeres and centromeres. This type of heterochromatin is mainly defined epigenetically by specific histone marks such as the di or tri-methylation of lysine 9 of histone H3 (H3K9me3) and the binding of the Heterochromatin protein 1a (HP1a)5,6. On the other hand, facultative heterochromatin localizes through the chromosome&#39;s arms and consists mainly of developmentally silenced genes7,8. Immunostaining of heterochromatin blocks in metaphase cells, or the observation of heterochromatin aggregates in interphase cells, has unveiled much light in the understanding of the formation and function of heterochromatic regions9.
The use of Drosophila as a model system has allowed the development of essential tools to study heterochromatin without the use of electron microscopy10. Since the description of position effect variegation and the discovery of heterochromatin-associated proteins, such as HP1a, and histone post-translational modifications, many groups have developed several immunohistochemical techniques that allow visualization of these heterochromatic regions10,11.
These techniques are based on the use of specific antibodies that recognize heterochromatin-associated proteins or histone marks. For every cell type and antibody, the fixation and permeabilization conditions must be determined empirically. Also, conditions may vary if additional mechanical processes such as squashing techniques are used. In this protocol, we describe the use of Drosophila salivary glands to study heterochromatic foci. Salivary glands have polytenized cells that contain more than 1,000 copies of the genome, thus providing an amplified view of most of the chromatin features, with the exception of satellite DNA and some heterochromatic regions which are under replicated. Nevertheless, heterochromatin regions are easily visualized in polytene chromosome preparations, but the squashing techniques may sometimes disrupt characteristic chromatin-bound complexes or the chromatin architecture. Therefore, immunolocalization of proteins in whole salivary gland tissue can surpass these undesired effects. We have used this protocol to detect several chromatin bound proteins, and we have demonstrated that this protocol combined with mutant Drosophila stocks can be used to study heterochromatin disruption12.

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	<tbody>
		<tr>
			<td>1.5 mL microcentrifuge tubes</td>
			<td>Axygen MCT-150-C</td>
			<td>11351904</td>
			<td>brand not critical</td>
		</tr>
		<tr>
			<td>16% formaldehyde</td>
			<td>Thermo Scientific</td>
			<td>28908</td>
			<td></td>
		</tr>
		<tr>
			<td>AF1 Citifluor</td>
			<td>Ted pella</td>
			<td>19470</td>
			<td>25 mL</td>
		</tr>
		<tr>
			<td>BSA, Molecular Biology Grade</td>
			<td>Roche</td>
			<td>10735078001</td>
			<td>brand not critical</td>
		</tr>
		<tr>
			<td>Complete, protease inhibitors Ultra EDTA-free 
			protease inhibitors</td>
			<td>Merck</td>
			<td>5892953001</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslip</td>
			<td>Corning</td>
			<td>CLS285022-200EA</td>
			<td>22x22, brand not critical</td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>Sigma</td>
			<td>d9779</td>
			<td>brand not critical</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma</td>
			<td>E5134</td>
			<td>brand not critical</td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td></td>
			<td></td>
			<td>brand not critical</td>
		</tr>
		<tr>
			<td>Glass slide</td>
			<td>Gold seal</td>
			<td>3011</td>
			<td>brand not critical</td>
		</tr>
		<tr>
			<td>H3BO3</td>
			<td>Baker</td>
			<td>0084-01</td>
			<td>brand not critical</td>
		</tr>
		<tr>
			<td>H3K9me3</td>
			<td>Abcam</td>
			<td>8889</td>
			<td></td>
		</tr>
		<tr>
			<td>HP1a</td>
			<td>Hybridoma Bank</td>
			<td>C1A9</td>
			<td>Product Form&#160;Concentrate 0.1 mL</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Baker</td>
			<td>3040-01</td>
			<td>brand not critical</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Baker</td>
			<td>9070-03</td>
			<td>brand not critical</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>71376</td>
			<td>brand not critical</td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td></td>
			<td></td>
			<td>brand not critical</td>
		</tr>
		<tr>
			<td>PIPES</td>
			<td></td>
			<td></td>
			<td>brand not critical</td>
		</tr>
		<tr>
			<td>Rotator</td>
			<td>Thermo Scientific</td>
			<td>13-687-12Q</td>
			<td>&#160;Labquake Tube Shaker</td>
		</tr>
		<tr>
			<td>Thermo Mixer C</td>
			<td>Eppendorf</td>
			<td>13527550</td>
			<td>SmartBlock 1.5 mL</td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Milipore</td>
			<td>648311</td>
			<td>brand not critical</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>T8787</td>
			<td>100 mL, brand not critical</td>
		</tr>
		<tr>
			<td>&#946;-mercaptoethanol</td>
			<td>Bio-Rad</td>
			<td>1610710</td>
			<td>25 mL, brand not critical</td>
		</tr>
	</tbody>
</table>

immunofluorescent staining for visualization of heterochromatin associated proteins in drosophila salivary glands

Visualization of heterochromatin aggregates by immunostaining can be challenging. Many mammalian components of chromatin are conserved in Drosophila melanogaster. Therefore, it is an excellent model to study heterochromatin formation and maintenance. Polytenized cells, such as the ones found in salivary glands of third instar D. melanogaster larvae, provide an excellent tool to observe the chromatin amplified nearly a thousand times and have allowed researchers to study changes in the distribution of heterochromatin in the nucleus. Although the observation of heterochromatin components can be carried out directly in polytene chromosome preparations, the localization of some proteins can be altered by the severity of the treatment. Therefore, the direct visualization of heterochromatin in cells complements this type of study. In this protocol, we describe the immunostaining techniques used for this tissue, the use of secondary fluorescent antibodies, and confocal microscopy to observe these heterochromatin aggregates with greater precision and detail.

Representative results of HP1a immunostaining in Drosophila salivary glands are shown in Figure 1. A positive result is to observe one focal point (Figure 1a) (heterochromatic aggregate or condensate). A negative result is no signal or a dispersed signal. Sometimes a double signal can be observed, that is, with a double point (Figure 1c), but it usually occurs in smaller quantities.
Data analysis...

Watch this Scientific Journal Video about Immunofluorescent Staining for Visualization of Heterochromatin Associated Proteins in Drosophila Salivary Glands at JoVE.com

Immunofluorescent Staining for Visualization of Heterochromatin Associated Proteins in Drosophila Salivary Glands

This protocol aims to visualize heterochromatin aggregates in Drosophila polytene cells.

Immunofluorescent Staining for Visualization of Heterochromatin Associated Proteins in Drosophila Salivary Glands

Visualization of heterochromatin aggregates by immunostaining can be challenging. Many mammalian components of chromatin are conserved in Drosophila ...

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