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 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's arms and consists mainly of developmentally silenced genes^7,8. 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 regions^10,11.

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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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 standard media.
3. Place the bottle with the flies in a controlled temperature incubator at 25 °C until the number of eggs laid is 50 (approximately 12 hours for the wild-type strain).
4. After the incubation time is over, remove the adults and transfer them to a new bottle to repeat the procedure. Let the embryos grow at 18 °C for 72 hours
   ​NOTE: For more about Drosophila stock maintenance conditions, see Tennessen & Thymmel¹³.

2. Larvae collection

1. For larvae collection choose the wandering larvae that do not have everted spiracles. After the eversion of the spiracles, the larva enters the prepupal stage, while retaining excellent polytene chromosomes suitable for analysis. Only after 12 hours do the cells of the salivary gland begin to prepare for programmed cell death^14,15.
2. Take fifteen 3° instar larvae and put them in a watch glass to wash them. Then transfer them to an ice-cold saline solution or PBS (1x PBS: 137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 2 mM KH₂PO₄, adjust pH to 7.4).
3. Dissect 15 to 30 pairs of salivary glands (or as many as possible in 30 minutes) in cold PBS with protease inhibitors under the stereoscopic microscope. Transfer the salivary glands to a 1.5 mL tube with ice-cold PBS.
4. Wash once with 1 mL of PBS plus protease inhibitors. Wait for the tissue to reach the bottom of the tube.
5. After the wash, remove the PBS with a 1000 µL pipette. Take care not to touch the tissue.
   1. Alternatively, dissect the salivary glands in 5 mL of PBS to eliminate the need for this washing step and proceed to step 3 by transferring the salivary glands to 0.5 mL of the Ruvkun fixing buffer described below.

3. Salivary gland tissue fixation

1. After removing the PBS from the last step, directly add 0.5 mL of 1x Ruvkun fixing buffer, with 50% methanol (add 0.5 mL of methanol) and 2% formaldehyde.
   ​NOTE: 2x Ruvkun solution is 160 mM KCl, 40 mM NaCl, 20 mM EGTA, 30 mM PIPES at pH 7.4.
2. Incubate for 2 hours at 4 °C with mild rotation.

4. Salivary gland tissue wash

1. Carry out one 5-minute rotation wash with 1 mL of Tris/Triton buffer (100 mM Tris pH 7.4, 1% Triton X-100 and 1 mM EDTA).
   ​NOTE: Wait for the tissue to reach the bottom of the tube.

5. Permeabilization

1. Incubate the salivary glands in 1 mL of Tris/Triton X-100 (the same as above). For some proteins it might be necessary to add 1% β-mercaptoethanol.
2. Incubate for 2 hours at 37 ° C with mild shaking (300 rpm).

6. Preservation step (optional)

NOTE: If not proceeding immediately to the incubation with the antibody, preserve the tissue as follows.

1. Wash with 1 mL of BO₃ buffer (0.01 M H₃BO₃ pH 9.2 + 0.01 M NaOH) and then incubate in BO₃/10 mM DTT at 37 ° C with mild shaking (300 rpm) for 15 minutes.
2. At the end of the incubation period, perform a wash with 1 mL of BO₃ buffer alone.
   NOTE: Wait for the tissue to reach the bottom of the tube.
3. Add 1 mL of PBS. Preserve the tissue in this solution at 4 °C for up to 72 hours and then proceed with the next step. This step is particularly helpful when working with different mutant strains that may present a delayed life cycle, so the immunodetection can be performed at the same time along with the controls.

7. Tissue blocking

1. Incubate the salivary glands in 1 mL of Buffer B (PBS + 0.1% BSA + 0.5% Triton X-100 + 1 mM EDTA) for 2 hours at room temperature with rotation.

8. Immunostaining

1. Remove all buffer B and add buffer A (PBS + 0.1% BSA) plus antibody of interest.
   ​NOTE: We use the HP1a C1A9c (concentrated antibody) from Hybridoma Bank up to 1:3000. When using the C1A9s (supernatant) we have tried from 1:100 to a 1:500 dilution and any dilution between this rank works well) overnight at 4 ᵒC with rotation. At this point it is important that the shaking does not raise bubbles which might damage the antibody.

9. Immunostaining washing

1. Perform 3 x 15-minute washes with buffer B under stirring at room temperature using 1 mL each time.
2. Transfer the glands to buffer B together with the secondary antibody coupled to a fluorophore for 2 hours under rotation at 4 ᵒC (secondary antibody Alexa fluor 568 Invitrogen were used 1:3000).
   1. Cover the tube with aluminum paper foil to protect the secondary antibody from the light.
3. Carry out 2 x 15-minute washes at room temperature while rotation with 1 mL of Buffer B.
4. Incubate with a DNA marker such as Sytox (take 2 µL of 5 mM stock and dissolve in 1 mL of Buffer B) or Hoechst (take 1 µL of 10 mg/mL stock and dissolve in 1 mL of Buffer B) for 10 minutes at room temperature with rotation.
5. Carry out one wash with Buffer B and once with PBS, each wash lasting 10 minutes while rotating at room temperature.
   ​NOTE: Remember to protect it from the light.

10. Imaging

1. Mount the salivary glands on a slide, making a pool with a coverslip.
2. Put the salivary glands in the middle of the pool and cover with AF1 citifluor to avoid the formation of bubbles extending the viscous liquid all over the place. Then seal all the sides with clear nail polish.
3. Observe under a fluorescence or confocal microscope. If the sample is not going to be observed on the same day, store away from light at 4 °C.
4. Use GraphPad Prism 6 to generate all graphs and statistical analyses.
5. Analyze the data from HP1a distribution in salivary glands using the Kruskal-Wallis test. Statistical significance was set at (p < 0.05*, < 0.01 **, < 0.001***, < 0.0001****).

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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...

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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,

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Materials

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