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Biology

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

Published: August 21st, 2021

DOI:

10.3791/62408

1Instituto de Biotecnología, Departamento de Genética del Desarrollo y Fisiología Molecular, Universidad Nacional Autónoma de México

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

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.

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 ....

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1. Third instar larvae culture

  1. 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.
    1. Afterward, let the media cool down to 60 °C and add propionic acid to a final concentration 0.01%. Let the bottle stand until the gelatin is formed.
  2. To op.......

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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.......

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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,

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We thank Marco Antonio Rosales Vega and Abel Segura for taking some of the confocal images, Carmen Muñoz for media preparation and Dr. Arturo Pimentel, M.C. Andrés 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 Tecnología (CONACyT) (A1-S-8239 to VV-G) and Programa de Apoyo a Proyectos de Investigación e Innovación Tecnológica (204915 and 200118 to VV-G)

....

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

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