Fluorescent in situ Hybridization Combined with Immunostaining on Polytene Chromosomes

Introduction

Polytene chromosomes are present in many larval tissues in Drosophila . They result from subsequent rounds of DNA replication not followed by cell division, and they represent a material of choice to determine by cytological methods whether a particular DNA sequence is associated with a protein of interest. They can be most easily prepared from salivary glands of third instar larvae, where the degree of polytenization is maximal.

We previously described a method that combined protein immunostaining with fluorescent in situ hybridization (FISH) in order to allow simultaneous visualization of a protein of interest and a specific DNA sequence (Dejardin and Cavalli, 2004; Lavrov et al ., 2004). Compared with previous methods, where FISH and immunostaining were performed in separate experiments, this approach has the great advantage of directly showing the co-localization of a protein with the locus of interest. However, this combination raises a number of technical difficulties. In some cases, the antibody immunostaining does not survive the FISH procedure. In the published protocol, we provided a set of solutions for this problem. In the worst situation, where the immunostaining signal can not preserved from degradation during FISH, one has to perform immunostaining, acquire many chromosome images under conditions in which it is possible to keep the record of the XY position of the microscope stage, then perform FISH, find the same chromosomes by repositioning the slide in the same XY coordinates as for immunostaining, acquire the FISH images, and finally superimpose the immunostaining and the FISH images using software such as Adobe® Photoshop® (Lavrov et al ., 2004). Although this procedure is straightforward, it is quite labor intensive.

Here, we present an adaptation of the protocol described earlier (Lavrov et al ., 2004). The main difference is that we reversed the order between the main steps. The FISH protocol is thus applied before immunostaining. This procedure is the simplest solution to the problems due to loss of immunostaining during the subsequent FISH procedure. For this reason, we recommend beginning with this protocol when analyzing the binding of your favorite protein on a specific DNA sequence present in polytene chromosomes.

However, it is possible that this protocol may not work for all epitopes (we have not yet encountered this problem), since the formaldehyde fixation of the chromosomes, performed before FISH, might not be sufficient to maintain the protein of interest anchored to the chromosomes throughout FISH. In this case, it is advisable to go back to the reverse procedure (Lavrov et al ., 2004; i.e. Immunostaining first, then FISH).

Below we describe a three-day protocol, with the DNA hybridization performed first (overnight between the first and the second day), and the incubation with the antibody against the protein of interest performed overnight between the second and the third days. The method consists of five parts: 1. Preparation of third instar larvae for extraction of salivary glands, 2. Chromosome squashes from salivary glands, 3. In situ DNA hybridization to polytene chromosomes, 4. Immunostaining with a specific primary antibody, 5. Immunodetection of both FISH and immuno signals using specific secondary antibodies.

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Procedure

Growth & collection of third instar larvae

1. Use bottles with rich medium (i.e. (i) 80g fresh yeast, 80g wheat flower, 11g agar, and 50ml Moldex in 1lt of water, or (ii) 8g agar, 18g dried yeast, 10g soybean meal, 7g molasses, 80g malt extract, 80g cornmeal and 6.3ml propionic acid in 1lt of water). Facultative: to improve growth conditions, you might add a large drop of live baker's yeast on top of the dried medium, and add a humidified filter paper with few water drops;
2. Let the flies lay eggs just to the point where larvae will hatch under uncrowded conditions (100 to 150 larvae/bottle);
3. Grow larvae at 18°C or higher temperature, but better results are obtained when larvae are raised at 18°C. Ideally, the humidity of the fly room should be maintained between 40-65%;
4. For salivary gland extractions, use third instar larvae that are slowly crawling and have not yet started to pupariate.

Extraction of salivary glands and chromosome squashes

1. Dissect one or two pairs of salivary glands in solution 1 (PBS). Best results are obtained by clipping the middle of the larval body with one pincet, and pulling the mouth hook outside the body with another pincet. Glands will come attached to the anterior parts of the larvae, and can then be easily dissected. Once dissected, get rid of most of the fat body cells without separating the two glands, if possible. Having the two glands still attached, will facilitate the following steps;
2. Transfer the glands directly with one pincet to a drop of solution 2 on a coverslip;
3. Fix the glands homogeneously by moving them with the pincet for 30 seconds in solution 2 (this time might need to be adjusted for individual antigens. For instance, very basic proteins such as histones require fixation for up to 5 minutes in solution 2);
4. Move glands into a droplet of solution 3 (43µl) on a coverslip and leave them for 2 minutes 30 seconds;
5. Take up the coverslip with a poly-L-lysine treated slide and quickly flip in order to bring the slide down and the coverslip up. Under the stereomicroscope, gently tap the coverslip with a pencil (2H) until cells are broken up. Hold the coverslip with one finger and extensively spread the chromosomes by (i) using the pencil, (ii) sliding back and forth or turning the coverslip. Check the spread under a phase contrast microscope. If the chromosomes are not spread enough, you can spread again using the method above. For a more extensive description of the chromosome spreading procedure, please refer to Lavrov et al ., 2004;
6. Remove the excess of fix solution by pressing slides (coverslip down) onto blotting paper with one finger;
7. Do not forget to mark the position of the coverslip on top of the slide. For this, we routinely use a diamond tip pen and make light incisions on the back of the slide, corresponding to two corners of the coverslip;
8. Place slides in liquid nitrogen. After freezing, quickly flick off the coverslip with a razor blade;
9. Wash slides two times for 15 minutes in PBS, slowly shaking the rack;
10. Proceed with the FISH experiment or keep the slides (up to 2-3 weeks) in 100% methanol.

 

In situ DNA hybridization of polytene chromosome spreads

Labeling and purification of the probe

DNA labeling : label 1µg of DNA using the BioNick™ Labeling System (Invitrogen™ LT) according to manufacturer instructions. Before stopping the reaction, check the nick-translation reaction by running 1/10 of the reaction on a 1% agarose gel. One should obtain a smear of biotin labeled fragments ranging from 500 to 200 nt. Stop the reaction according manufacturer instructions.

DNA Purification : two alternative procedures (see note 1)

(i) EtOH precipitate the DNA: add 3M sodium acetate to the reaction mixture (1/10 of total volume) and carry out ethanol precipitation by adding 2 volumes of cold 96% ethanol (EtOH), and incubating overnight at -200°C. After centrifugation (14.000 rpm, 4°C, 30 minutes), rinse the biotynilated DNA pellet once with 70% EtOH, dry it and dissolve it in 20µl of TE. Mix the solution of biotynilated DNA with 200µl of hybridization buffer. The mixture is stable at -20°C.

(ii) Use a PCR purification kit (QIAGEN®). Add 500µl of binding buffer PB, and pass the mixture though a column. Wash with EtOH containing buffer PE, and elute with bidistilled water. Concentrate the probe up to 10-20µl by using a speed-vac, and then mix with 100-200µl of hybridization buffer.

DNA hybridization on polytene chromosomes

1. Put slides into 2XSSC at room temperature (RT), and then put the rack with slides into a 700°C waterbath for 45 minutes;
2. Dehydrate slides by passing through 70% EtOH (2x5 minutes) and 96% EtOH (2x5 minutes);
3. Denature chromosomal DNA by incubation in 0.07M NaOH for 10 minutes. After denaturation, wash the slides in 2xSSC (1 minute+1 minute+5 minutes) and dehydrate as above;
4. Denature the hybridization mixture with biotynilated DNA (one aliquot for each slide) by incubating 5 minutes at 95°C, then snap cooling in ice. Pre-warm to 37°C and load 12µl of the mixture on the slide. Cover with a coverslip, avoiding formation of air bubbles. Carry out hybridization overnight in a humid chamber at 37°C;
5. After overnight hybridization, remove coverslips and wash slides in 2XSSC 3x5 minutes at 42°C and 1x5 minutes at RT.

Immunostaining

1. Wash slides 1x15 minutes in PBS;
2. Block for 1 hour in blocking solution at RT;
3. Add 20µl of primary antibodies diluted in blocking solution to each slide (dilutions need to be adjusted for each individual primary antibody, i.e. for most rabbit polyclonal antibodies, dilutions are usually between 1:30 and 1:500). Cover with coverslip and incubate overnight at 40°C in a humid chamber;
4. After the overnight incubation, rinse 2x5 minutes in PBS 5 minutes at RT;
5. Wash 15 minutes in PBS, 300mM NaCl, 0.2% NP40, 0.2% Tween20. Shake rack thoroughly;
6. Wash 15 minutes in PBS, 400mM NaCl, 0.2% NP40, 0.2% Tween20. Shake rack thoroughly;
7. Rinse in PBS 5 minutes. (See note 2)

Immunodetection

Detection of the FISH signal

1. Add 20µl of the diluted FITC-anti-Biotin antibody (1/500e in PBS + 4% BSA) on the slide. Cover with a coverslip and incubate for 1 hour at RT in a humid chamber in the dark. From now on, perform all incubations in the dark;
2. Wash in PBS at room temperature for 5 minutes.

Detection of the Immunostaining

1. Add 20µl of the diluted Cy3-Anti-Rabbit secondary antibody (1/700e in blocking solution + 2% normal goat serum). Cover with a coverslip and incubate for 45 minutes at RT in humid chamber;
2. Rinse in PBS for 5 minutes;
3. Wash 15 minutes in PBS, 300mM NaCl, 0.2% NP40, 0.2% Tween20. Shake rack thoroughly;
4. Wash 15 minutes in PBS, 400mM NaCl, 0.2% NP40, 0.2% Tween20. Shake rack thoroughly;
5. Rinse in PBS for 5 minutes.

Cytology

1. Counterstain the DNA with DAPI (dilute the stock solution of DAPI 1:200 in PBS freshly) for 10 minutes at RT;
2. Wash 5 minutes in PBS;
3. Mount the chromosomes in 40µl of mowiol stock solution (or commercially available mounting media);
4. Analyze the slides under a fluorescent microscope equipped with a 40X objective. For better resolution, one might use 63X or 100X oil objectives. An exemple of FISH-immunostaining is shown in Figure 1. For a detailed description of image processing, see (Lavrov et al ., 2004).

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Materials & Reagents

PBS	Solution 1 - the pH of the PBS solution is 7.5
solution 2	3.7% paraformaldehyde, 1% Triton X-100, in PBS (37% p-formaldehyde stock solution: weigh 1.85g of p-formaldehyde powder. Add in H2 O (final volume of 5ml), add 70µl 1 N KOH, dissolve by boiling).
solution 3	3.7% p-formaldehyde, 50% acetic acid, in H2 O
mowiol stock solution	Add 2.4 g Mowiol to 6g Glycerol and 6ml H2 O. Mix for 3 hours, add 12ml 0.2M Tris-HCl pH 8.5 and incubate 30 minutes at 60°C with mixing. Insoluble material is pelleted by centrifugation for 15 minutes at 5000xg. Add DABCO (Diazabicyclo(2.2.2.) octane. Merck® #803456) to final 2.5% to the solution as anti-bleaching agent. Make 500µl aliquots and store at -20°C Alternatively, one could use commercially available mounting media such as Vectashield™ (Vector®) or ProLong® antifade (Molecular Probes™)
blocking solution	BSA 3% 0.2% (w/v) NP40 0.2% (w/v) Tween 20 10% non fat dry milk (recipe for 500ml: 440ml PBS 1X, 15g BSA, 1g NP40, 1g Tween 20, 50g non fat dry milk. Store 50ml aliquots at -20°C) PBS 1X 4% BSA
hybridization buffer	2XSSC 10% dextransulfat 50% formamide 0.8mg/ml (0.08%) salmon sperm DNA
DAPI	(4'6' Diamidino-2-phenylindole) stock solution at 0.1mg/ml in 180mM Tris-HCl, pH 7.5