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

Summary

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

Abstract

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.

Introduction

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 stability^2,3,4.

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 ....

Protocol

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 optimize the 3ᵒ instar larvae culture, first collect 5-to-10-day old adults and place 50 (25 males and 25 females) in a broad neck bottle of stand....

Results

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.......

Discussion

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,

Materials

List of materials used in this article

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