Name: Preparation of Drosophila Polytene Chromosome Squashes for Antibody Labeling (Video) | JoVE Journal
Uploaded: 2010-02-09T17:00:00.000+00:00
Duration: 6 min 37 s
Description: Learn the squash technique for preparing Drosophila polytene chromosomes for antibody labeling. This protocol demonstrates dissecting salivary glands, fixation, and microscopy to study gene expression.

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5 minutes

We thank Ms. V. Lephart for maintenance of fly stocks This work was supported by National Institutes for Health grant (GM62916) and National Science Foundation grant (MCB0817107).

The following protocol for polytene chromosome squash preparation is adapted from the procedure described in Johansen et al. (2009).
1. Culture of third instar Drosophila larvae
In order to obtain optimal polytene chromosomes for high quality squash preparations, uncrowded culturing conditions are essential (i.e., place around 20 egg-laying female flies in a standard 4" fly bottle and change to a new bottle each day). Select the fattest individuals from the first crop of climbing 3rd instar larvae while they are still wandering but just prior to pupation. We routinely culture at 21°C but 18°C will yield fatter chromosomes that may be more suitable for some purposes such as for example when band/interband regions need to be visualized at high resolution.
2. Polytene squash materials
<ul>
<li>Drummond dissection forceps (2)</li>
<li>Petri dishes (60 x 15 mm)</li>
	<li>Frosted microscope slides (Fisher No. 12-544-3) (poly-lysine-coated)</li>
	<li>22 x 22 mm No. 15 cover slips (Fisher No. 12-520B) (coated with Sigmacote; Sigma #SL2)</li>
	<li>22 x 40 mm No. 15 cover slips (Fisher No. 12-530B)</li>
	<li>Kim-wipes</li>
	<li>Phase contrast microscope with 20X objective</li>
	<li>Small Dewar (e.g., vacuum flask or thermos bottle)</li>
	<li>Long forceps</li>
	<li>Razor blades</li>
	<li>Coplin Jar</li>
	<li>Rubber-Maid or Tupperware tray (or equivalent sealable tray)</li>
	<li>Parafilm (cut into 22 mm squares)</li>
	<li>175 g weights</li>
</ul>
3. Fixatives and solutions
<ol>
	<li>5X formaldehyde stock. Prepare a fresh solution of 0.74 g. paraformaldehyde in 4.0 ml of dH2O and with 28 μl of 1N KOH. Warm to 65°C to dissolve the paraformaldehyde and then store on ice.</li>
	<li>Fixative 1. Prepare a fresh solution of 0.5 ml 10X PBS, 50μl Triton X-100, 3.45 ml dH2O and 1.0 ml from a 5X formaldehyde stock. Warm to disperse the Triton X-100 and use within 1 hour.</li>
	<li>Fixative 2. Prepare a fresh solution of 1.5 ml dH2O, 2.5 ml Glacial Acetic Acid, and 1.0 ml from a 5X formaldehyde stock and use within 1 hour.</li>
	<li>Lactoacetic acid solution. Prepare a solution of 1 ml lactic acid, 2 ml dH2O, and 3 ml acetic acid.</li>
</ol>
4. Polytene chromosome squash preparation:
<ol>
	<li>Rinse larvae with water and transfer to PBS in a tissue culture dish for dissection.</li>
	<li>Grasp the tip of the mouth hooks with one pair of forceps, hold the body about 2/3 of the way down with the other pair, and pull on the mouth hooks so the salivary glands are exposed. Separate the salivary glands from the brain and eye-antennal discs, and dissect away the fat body and any other associated tissues from the glands.</li>
	<li>Add 200 microliters of Fixative 1 to the first well of a two well depression slide and 200-300 microliters of Fixative 2 to well two. Transfer one pair of salivary glands at a time to Fixative 1 in well one and incubate for the amount of time required for the target epitope, usually about 1-2 minutes. (Note: some epitopes, such as for most of the histone modifications, may require 5 minutes or longer fixation.)</li>
	<li>Using forceps transfer the salivary glands to Fixative 2 in well 2 and incubate for two minutes.</li>
	<li>Transfer the glands to 10-30 μl of Lactoacetic acid solution on a clean Sigmacoted coverslip. Gently lower a polylysine-coated microscope slide onto the coverslip and, without applying vertical pressure, pick up the coverslip so the glands are between the slide and the coverslip. Immediately facilitate cell lysis and chromosomal spreading by carefully grasping the coverslip with forceps on one edge and gently moving it slightly back and forth, trying to minimize any vertical pressure on the tissue. Alternatively use the eraser side of a pencil or even a fingertip to gently move the coverslip back and forth. Note that any delay in moving the coverslip back and forth is likely to diminish spreading of the chromosomal arms as they will tend to become more rigid upon exposure to the Lactoacetic acid solution. Clouding of the solution is often a good indication of cell separation. Gently tapping the coverslip obliquely (to avoid vertical pressure) with the eraser side of a pencil a few times may also assist in chromosomal spreading.</li>
	<li>Immediately examine the tissue under a phase contrast microscope with a 20 or 40X objective to determine whether the chromosomes are well-spread.</li>
	<li>When the chromosomal spreading is sufficient, set the slide with the coverslip side down on a stack of clean Kim-wipes. Place a second Kim-wipe on top and flatten chromosomes by placing your thumb over where the coverslip is positioned and pressing firmly, avoiding any horizontal movement of the coverslip that would shear the chromosomes.</li>
	<li>Examine the slide again under the microscope to determine if the preparation is suitable for the intended purpose. If chromosomes have moved significantly upon squashing, use less volume of Lactoacetic acid solution in your subsequent preparations. Repeat steps 4.1-4.8 until a sufficient number of suitable slides have been obtained. Place 175 g weights upon the coverslip of the slides.</li>
	<li>Fill a small Dewar (e.g., thermos bottle) with liquid nitrogen. Using a long forceps, dip slide into liquid N2 until the boiling stops, remove slide, and immediately use the edge of a clean razor blade at one corner to flip off the coverslip. If slide will be used immediately, place in a Coplin jar with PBS at 4°C. Otherwise collect the slide into a Coplin jar filled with 95% ethanol. Examine the coverslip once the frost has sublimed away to confirm that the tissue adhered to the slide and did not remain with the coverslip. Repeat with the remainder of the slides until all slides are stored in either PBS (for use within several hours) or ethanol (for longer term storage).</li>
</ol>
Representative Results
The first critical step for obtaining high quality polytene squash preparation is to grow fat larvae with big salivary gland nuclei. The second is good technique with the spreading procedure, which may take some practice. One tip for improved spreading success is to find the minimal volume of lactoacetic acid solution during the squashing step. This will promote sufficient spreading of chromosomes without generating excessive streaming forces that can wash chromosome arms away. It is also worth noting that any delay in moving the coverslip back and forth will reduce spreading of the chromosomal arms as they become more rigid when exposed to the Lactoacetic acid solution.
If everything goes well, there should be numerous well spread polytene chromosomes. Figure 1 shows an example of such a preparation, double labeled with a marker for interband regions in green and with a dye that stains the banded regions in blue. If insufficient spreading is obtained, the chromosomes will look like little balls, as shown in Figure 2. On the other hand, if too much spreading has occurred, the chomosomes will be too thin and extended or in some cases fragmented into small pieces as shown in Figure 3.
<img src="/files/ftp_upload/1748/1748fig1.jpg" alt="Chromosome structure, fluorescence microscopy image, illustrating DNA organization in cellular biology." data-altupdatedat="1788235205000" data-alt="Figure 1"> Figure 1. Polytene squash preparation double labeled with antibody to the JIL-1 histone H3S10 kinase (in red) and Hoechst (blue).
<img src="/files/ftp_upload/1748/1748fig2.jpg" alt="Chromosome structure, fluorescence microscopy, DNA analysis, genetic research, cell nucleus study." data-altupdatedat="1788235205000" data-alt="Figure 2"> Figure 2. Polytene squash with insufficient spreading.
<img src="/files/ftp_upload/1748/1748fig3.jpg" alt="Neuronal structure microscope image, synaptic connection diagram for neuroscience research analysis." data-altupdatedat="1788235205000" data-alt="Figure 3"> Figure 3. Polytene squash with too much spreading.

<ol><li>Cai, W., Bao, X., Deng, H., Jin, Y., Girton, J., Johansen, J., Johansen, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((RNA+polymerase+II-mediated+transcription+at+active+loci+does+not+require+histone+H3S10+phosphorylation+in+Drosophila%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">RNA polymerase II-mediated transcription at active loci does not require histone H3S10 phosphorylation in Drosophila.</a> Development. 135, 2917-2925 (2008).</li>
<li>DiMario, P., Rosby, R., Cui, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Direct+visualization+of+GFP-fusion+proteins+on+polytene+chromosomes%5BTitle%5D)+AND+%22Dros.+Inf.+Serv%22%5BJournal%5D)">Direct visualization of GFP-fusion proteins on polytene chromosomes.</a> Dros. Inf. Serv. 89, 115-118 (2006).</li>
<li>Johansen, K. M., Cai, W., Deng, H., Bao, X., Zhang, W., Girton, J., Johansen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Methods+for+studying+transcription+and+epigenetic+chromatin+modification+in+Drosophila+polytene+chromosome+squash+preparations+using+antibodies%5BTitle%5D)+AND+%22Methods%22%5BJournal%5D)">Methods for studying transcription and epigenetic chromatin modification in Drosophila polytene chromosome squash preparations using antibodies.</a> Methods. 48, 387-397 (2009).</li>
</ol>

The inclusion of acetic and lactic acids in conventional squash fixation protocols facilitates both interband resolution and chromosomal arm spreading but unfortunately some epitopes do not survive this treatment. An example of such an epitope is H3S10ph (Cai et al., 2008). Since acid treatment also has the disadvantage that it quenches the inherent fluorescence of GFP-tagged proteins, DiMario et al. (2006) recently developed a formaldehyde-based "acid-free squash technique" that allow for direct visual...

preparation of drosophila polytene chromosome squashes for antibody labeling

Preparation of Drosophila Polytene Chromosome Squashes for Antibody Labeling

Drosophila has long been a favorite model system for studying the relationship between chromatin structure and gene regulation due to the cytological advantages provided by the giant salivary gland polytene chromosomes of third instar larvae.  In this tissue the chromosomes undergo many rounds of replication in the absence of cell division giving rise to approximately 1000 copies.  The DNA remains aligned after each replicative cycle resulting in greatly enlarged chromosomes that provide a unique opportunity to correlate chromatin morphology with the localization of specific proteins.  Consequently, there has been a high level of interest in defining the epigenetic modifications present at different genes and at different stages of the transcription process. An important tool for such studies is the labeling of polytene chromosomes with antibodies to the enzyme, transcription factor, or histone modification of interest. This video protocol illustrates the squash technique used in the Johansen laboratory to prepare Drosophila polytene chromosomes for antibody labeling.

This video protocol illustrates the squash technique used in the Johansen laboratory to prepare Drosophila polytene chromosomes for antibody labeling.

Drosophila has long been a favorite model system for studying the relationship between chromatin structure and gene regulation due to the cytological ...

This procedure begins with culturing fat crawling drosophila third in star larvae in a fly bottle. Using two pairs of forceps, the salivary glands are dissected away from the larvae. The salivary glands are then successfully fixed in two different fixatives in a two well depression slide.Finally, the glands are squashed in a lacto acetic acid solution between a cover slip and a microscope slide. Hi, I'm Dr.Kristen Johansen in the Department of Biochemistry, biophysics and molecular biology at Iowa State University. Hi, I am TA graduate student in the Johansen's lab.Today we're gonna show you a procedure for making drosophila polytan chromosome squash preparations suitable for antibody labeling. We use this procedure in our laboratory to study epigenetic histo modifications and the relationship to chromatin structure and gene expression. So let's get started.In order to obtain optimal polytan chromosomes for high quality squash preparations, uncrowded culturing conditions are essential. Place around 20 egg laying female flies in a standard four inch fly bottle and change to a new bottle each day. Culturing at 21 degrees Celsius is fine for most purposes, but incubation at 18 degrees Celsius will yield fat or chromosomes that may be more suitable for some experiments, such as when banned interband regions need to be visualized at high resolution.Select the fattest individuals from the first crop of climbing third in star larvae. While they are still wandering, but just prior to pation, the larvae are easily visible on the side of the bottle above the food. Use forceps to place larvae on a tissue culture dish.For each round of squashing, rinse two to three of the larvae together with water before transferring them to a clean dish of PBS for dissection. The PBS should cover the larvae, but time is not of the essence. Since the larvae can survive under these conditions for hours, place the dish of larvae in PBS on the stage of a dissecting microscope with one pair of forceps.Grasp the tip of the mouth hooks while holding the body. About two thirds of the way down with a second pair, pull on the mouth hooks with the forceps to expose the salivary glands. Separate the salivary glands from the brain and eye and tenal discs and dissect away the fat body and any other associated tissues.Add 200 microliters of fixative one to the first well of a two well depression slide and 200 to 300 microliters of fixative. Two to well two. Transfer one pair of salivary glands at a time to fix it of one in well one and incubate for the amount of time required for the target epitope usually about one to two minutes using forceps.Transfer the salivary glands to fix it of two in, well two and incubate for two minutes Using forceps, transfer the glands to a clean sigma coated cover slip with 10 to 30 microliters of freshly prepared lacto acetic acid solution. Gently lower a polylysine coated microscope. Slide onto the cover slip.Carefully lift the slide using the bonding of the liquid to the glass to pick up the cover slip without applying vertical pressure. To the preparation to spread the chromosomes immediately and carefully grasp the edge of the cover slip with forceps and gently move it slightly back and forth. Again, minimizing vertical pressure on the tissue, the wiggling of the cover slip is important for adequately separating the individual cells of the salivary gland.Gently tapping the cover slip obliquely with the eraser side of a pencil may also assist in spreading clouding of the solution is often a good indication of cell separation. Promptly examine the tissue under a phase contrast microscope to check that the chromosomes are well spread as indicated by clearly separated and extended chromosome arms. If the chromosomes do not appear, well spread the first time.It is possible to repeat the gentle tapping of the cover slip until sufficient spreading is obtained. Set the slide cover slip side down on a stack of clean Kim wipes. Place a clean wipe on top of the preparation.Place a thumb over the position of the cover slip and firmly press straight down. Avoid horizontal movement, which would shear the chromosomes. Reexamine the slide under the microscope to determine suitability for the current experiment.Repeat the dissection and chromosome squash preparation to obtain a sufficient number of slides. Place a 175 gram weight on the cover slip of the slides using long forceps. Dip a prepared slide into a small doer of liquid nitrogen and hold it there until the boiling stops.Remove the slide and immediately use the edge of a clean razor blade under the corner of the cover slip to flip it off. Collect the slide into a coplan jar filled with cold PBS for immediate use, or filled with 95%ethanol for longer storage. Once the frost has sublimed away, examine the cover slip to confirm that the tissue remained with the slide.The first critical step for obtaining high quality INE squash preparation is to grow fat larvae with big salivary gland nuclei. The second is good technique with the spreading procedure, which may take some practice. One tip for improving spreading success is to find the minimal volume of lacto acetic acid solution during the squashing step.This will promote sufficient spreading of chromosomes without generating excessive streaming forces that can wash chromosome arms away. It is also worth noting that any delay in moving the cover slip back and forth will reduce spreading of the chromosomal arms as they become more rigid when exposed to the lacto acetic acid solution. If everything goes well on each slide, there should be numerous well spread polytan chromosomes.Here we show an example of such a preparation double labeled with a marker for interband regions in red and with a dye which stains the banded regions in blue. If insufficient spreading is obtained, the chromosomes will look like little balls as shown here. On the other hand, if too much spreading has occurred, the chromosomes will be too thin and extended or in some cases fragmented into small pieces as shown here.So we've just shown you how to make drosophila poly team chromosome squash preparations When doing this procedure. It is important to remember to start with fat la. So that's it.Thanks for watching and good luck with your experiments.

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