Name: dissection and staining of drosophila pupal ovaries
Uploaded: 2018-03-02T14:30:00
Description: Watch this Scientific Journal Video about Dissection and Staining of Drosophila Pupal Ovaries at JoVE.com

This research was supported by the National Institutes of Health (RO1 GM079351 to D.K.). We thank Dorothea Godt for her helpful advice on pupal ovary dissections based on her original protocol. We also thank Amy Reilein for her assistance and comments on the manuscript.

1. EggLaying
<ol>
	<li>Combine approximately ten male and fifteen female adult Drosophila flies of the desired genotype in a vial of normal rich fly food supplemented with yeast. To avoid overcrowding the vial, allow mated females to lay eggs for no longer than 2&#8211;4 h14.</li>
	<li>Transfer the adults from the vial into a new vial by tapping the vial opening against a different vial with fly food. Allow the eggs to develop into larvae at room temperature for 3&#8211;4 days.</li>
</o...

<ol>
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The most critical and difficult step of this protocol involves the preparation of pupal ovaries prior to fixation. To ensure that the ovaries, small and buried by fat body cells inside the pupal abdomen, are stained sufficiently with antibodies, it is important to not only tear a large opening in the abdominal sack with forceps, but also extract the fat body cells that obstruct the ovaries from the antibodies. Successful execution of this step requires application of subtle pressure on the Pasteur pipet bulb while washin...

Stem cell research using Drosophila ovaries has widely expanded since the first documentation of a stem cell niche1,2,3,4. Following the development of lineage tracing genetic tools, Drosophila ovary dissections have been commonly used to study stem cell lineages and signaling pathways that regulate stem cell maintenance, proliferation, and fate in the stem cell niche. Knowledge of these signaling pathways may yield insights into potential causes of cancers that originate from aberrant stem cell activity5,6,7. It has also recently been shown that somatic stem cells in the Drosophila ovary, known as follicle stem cells (FSCs), strongly resemble mammalian intestinal stem cells in many aspects of their organization8. For this reason, Drosophila ovaries are a highly useful model system for studying stem cell behavior.
While larval and adult ovaries offer clues to early stem cell development and final stem cell organization in the niche, respectively, the pupal ovary is an intermediate structure in which the germline and somatic cells reorganize and establish their identities9,10. Though several studies have examined aspects of tissue development in the pupal ovary10,11,12,13, questions remain regarding the differentiation and spatial organization of ovarian cell types during pupal development. In particular, the specification of FSCs occurs during this period. This protocol outlines a method for dissecting and staining pupal ovaries at desired time points&#8212;a technique that can be used in time course experiments that analyze pupal ovary development in detail from the larval to the adult stage.
To account for the small size, translucence, and inaccessibility of the pupal ovary within the pupal abdomen, this protocol utilizes tools such as a custom-made thin-tipped Pasteur pipet to remove abdominal fat body tissue obstructing antibody access to the ovaries. A clear, chambered coverglass used during the antibody staining offers greater visibility of the pupae and a gentler platform for rocking the ovaries on a &#34;Nutator.&#34; Based on a protocol for larval ovary dissections by Maimon and Gilboa14, a relatively high concentration of Triton X-100 has been employed in the initial steps of the staining procedure to maximize cell membrane permeabilization and antibody access to the ovarian cells.

<table>
	<tbody>
		<tr>
			<td>Dumont #5 Forceps, biology</td>
			<td>Fine Scientific Tools</td>
			<td>11252-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Nunc Lab-Tek Chambered Coverglass</td>
			<td>Thermo Fisher Scientific</td>
			<td>155383</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection microscope</td>
			<td>Nikon</td>
			<td>SMZ-10A</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal Microscope</td>
			<td>Carl Zeiss</td>
			<td>LSM 700</td>
			<td></td>
		</tr>
		<tr>
			<td>Analysis software</td>
			<td>Carl Zeiss</td>
			<td>Zen</td>
			<td></td>
		</tr>
		<tr>
			<td>9 Depression Glass Spot Plates</td>
			<td>Pyrex</td>
			<td>7220-85</td>
			<td></td>
		</tr>
		<tr>
			<td>Pasteur pipet</td>
			<td>Fisher Scientific</td>
			<td>13-678-6B</td>
			<td></td>
		</tr>
		<tr>
			<td>Pasteur pipet bulb</td>
			<td>Various vendors</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bunsen burner</td>
			<td>Various vendors</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherfinest Premium Frosted Microscope Slides</td>
			<td>Thermo Fisher Scientific</td>
			<td>12-544-2</td>
			<td></td>
		</tr>
		<tr>
			<td>22 x 22 mm glass coverslips No 1</td>
			<td>VWR</td>
			<td>48366-067</td>
			<td></td>
		</tr>
		<tr>
			<td>Dapi Fluoromount-G</td>
			<td>SouthernBiotech</td>
			<td>0100-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Double-sided tape</td>
			<td>Scotch</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nutator</td>
			<td>Clay Adams</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fine brush #0, #3-#5</td>
			<td>Various vendors</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gilson Pipetman Starter Kit</td>
			<td>Thomas Scientific</td>
			<td>F167300</td>
			<td>Contains p20, p200, p1000 pipettors</td>
		</tr>
		<tr>
			<td>16% Paraformaldehyde</td>
			<td>Electron Microscopy Sciences</td>
			<td>15710</td>
			<td>Dilute to 4% paraformaldehyde in 1x PBS</td>
		</tr>
		<tr>
			<td>Triton</td>
			<td>Sigma-Aldrich</td>
			<td>9002-93-1</td>
			<td></td>
		</tr>
		<tr>
			<td>10x PBS</td>
			<td>Ambion</td>
			<td>AM9624</td>
			<td>Dilute to 1x PBS</td>
		</tr>
		<tr>
			<td>Normal Goat Serum</td>
			<td>Jackson ImmunoResearch</td>
			<td>5000121</td>
			<td>Dilute to 10% normal goat serum in PBST with 0.5% Triton concentration</td>
		</tr>
		<tr>
			<td>Primary antibodies (in protocol: 7G10 anti-Fasciclin III diluted 1:250, rabbit anti-phosphohistone H3 diluted 1:1000)</td>
			<td>Various vendors (in protocol: Developmental Studies Hybridoma Bank, Millipore)</td>
			<td></td>
			<td>Dilute in PBST with 0.5% Triton concentration</td>
		</tr>
		<tr>
			<td>Secondary antibodies (in protocol: Alexa-546, FITC-conjugated anti-rabbit serum)</td>
			<td>Various vendors (in protocol: Molecular Probes, Jackson ImmunoResearch Laboratories, Inc.)</td>
			<td></td>
			<td>Dilute in PBST with 0.5% Triton concentration</td>
		</tr>
		<tr>
			<td>Fly vials</td>
			<td>Denville Scientific</td>
			<td>V9406</td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton Balls, For Wide Vials</td>
			<td>Genesee Scientific</td>
			<td>51-102W</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast, Bakers Dried Active</td>
			<td>MP Biomedicals</td>
			<td>101400</td>
			<td></td>
		</tr>
		<tr>
			<td>Fly food</td>
			<td>Produced in laboratory</td>
			<td></td>
			<td>Mixture of water, brewer&#39;s yeast, cornmeal, molasses, agar, EtOH, penicillin, methyl 4-hydrobenzoate, and propionic acid</td>
		</tr>
		<tr>
			<td>Male and female Drosophila flies (genotype used in protocol: yw; P[Fz3-RFP, w+]/TM2)</td>
			<td>Bloomington Drosophila Stock Center</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

dissection and staining of drosophila pupal ovaries

Unlike adult Drosophila ovaries, pupal ovaries are relatively difficult to access and examine due to their small size, translucence, and encasing within a pupal case. The challenge of dissecting pupal ovaries also lies in their physical location within the pupa: the ovaries are surrounded by fat body cells inside the pupal abdomen, and these fat cells must be removed to allow for proper antibody staining. To overcome these challenges, this protocol utilizes customized Pasteur pipets to extract fat body cells from the pupal abdomen. Moreover, a chambered coverglass is used in place of a microcentrifuge tube during the staining process to improve visibility of the pupae. However, despite these and other advantages of the tools used in this protocol, successful execution of these techniques may still involve several days of practice due to the small size of pupal ovaries. The techniques outlined in this protocol could be applied to time course experiments in which ovaries are analyzed at various stages of pupal development.

Successful execution of this procedure should result in clear antibody staining that reveals the structure and cellular organization of a Drosophila pupal ovary. Immunohistochemistry outlined in this protocol can be used to identify cell types commonly stained in larval and adult ovaries. Cells of the pupal stalk derived from swarm cells18 (outlined by Fasciclin III in white) are shown in Figure 3. In addition to highlighting ...

Watch this Scientific Journal Video about Dissection and Staining of Drosophila Pupal Ovaries at JoVE.com

Dissection and Staining of Drosophila Pupal Ovaries

The Drosophila ovary is an excellent model system for studying stem cell niche development. Though methods for dissecting larval and adult ovaries have been published, pupal ovary dissections require different techniques that have not been published in detail. Here we outline a protocol for dissecting, staining, and mounting pupal ovaries.

Dissection and Staining of Drosophila Pupal Ovaries

Unlike adult Drosophila ovaries, pupal ovaries are relatively difficult to access and examine due to their small size, translucence, and encasing within a ...

The overall goal of this procedure is to dissect and stain drosophila pupal ovaries. This method can help answer key questions in development of biology, such as those pertaining to stem cell differentiation and tissue development in the model organism drosophila. The main advantage of this technique is that customizable tools are used to accommodate the small size and translucent nature of the pupal ovaries, which are otherwise difficult to isolate.We had the idea to use his method when we conducted time course experiments, to track stem cell division in the ovary during pupal drosophila development. To begin the protocol, combine 10 male and 15 female adult drosophila flies, in a vial of normal rich fly food supplemented with yeast. Allow the eggs to develop into larvae at room temperature for three to four days.After three to four days, use a moist fine brush with soft bristles to make a rolling movement with the brush along the wall of the vial, to transfer wandering third instar larvae, from the vial to a glass well, filled to the brim with 1X phosphate buffered saline or PBS. Wash the food debris off the larvae, by transferring them to another well filled with 1X PBS. Separate the male and female larvae with forceps.Identify the male larvae by a pair of large round and translucent testes embedded in the fat body on the lateral side two-thirds down from its anterior end. Then collect at least 10 females from the well and place the female larvae into a separate well, filled with 1X PBS. Use forceps to transfer the female larvae gently into a new vial of fresh fly food supplemented with yeast.Place the vial with the female larvae in a dark location to facilitate pupariation. Throughout the day, monitor the larvae for pupariation. As each larva lobalizes and develops into a pre-pupa, circle the pre-pupa against the vial and record the approximate time when it first forms into a pre-pupa.Melt the glass tip of a pester pipette over a Bunsen burner. As the glass melts, use forceps to pull the tip horizontally away from the rest of the pipette to form a thinner tip. After cooling, break off a small portion of the pipette tip to form a neat circular opening.Attach a bulb to the other end of the pipette and load the pipette with 1X PBS. To harvest the pupae glued to the wall of the vial apply a small drop of water along the contact zone between pupa and vial. After allowing the protein glue to dissolve for one to two minutes, gently lift the pupa off the wall with a moist fine brush.Transfer a single pupa per well filled with 1X PBS. While grasping the posterior end of the pupa with one pair of forceps, carefully tear the anterior portion of the pupal case with another pair until the head of the pupa is visible. Grip the anterior most tip of the pupal head with forceps, and gently pull the pupa out of its pupal case.Next separate and discard the anterior half of the pupa from the posterior half until only the abdominal sac remains. Grasp the abdominal sac against the bottom of the well with forceps in one hand, while holding the thin glass pipette filled with 1X PBS in another hand. Aim the thin pipette tip towards the opening of the sac and slowly pipette 1X PBS into the abdomen to wash away the fat body cells surrounding the pupal ovaries.Wash away the fat body cells until at least two thirds of the fat body is gone, or until the ovaries are visible near the opening of the abdominal sac. If the ovaries are not visible inside the well, they should've remained in the abdomen. If the ovaries have come out, place a new pupa in the well and repeat this step.Transfer the abdomen into a clear covered glass chamber filled with fixation buffer, and place the lid on top. Dissect more ovaries than needed to ensure enough ovaries would stand at the staining and mounting process. Throughout the staining process, use forceps to gently push down any floating ovaries to the bottom of the covered glass well to ensure that the ovaries are completely immersed in antibody solution.Place strips of double-sided tape on to the flat surface of the new titer and then place the chambered cover glass containing the ovaries and fixation buffer on the adhesive surface. Incubate the ovaries in fixation buffer for 15 minutes at room temperature. Next, rinse the ovaries three times in 1X PBS with 1%Triton X-100 or 1%PBST for five, 10, and 45 minutes respectively, to allow thorough permeabilization.Lock the ovaries in 10%normal goat serum in 0.5%PBST for 30 minutes. Then incubate the ovaries overnight in 600 microliters of primary antibody in 0.5%PBST at 4%Celsius. The next day rinse the ovaries three times for five minutes each in 0.5%PBST.Incubate the ovaries for two hours in 600 microliters of secondary antibody diluted in 0.5%PBST at room temperature. After incubation, rinse the ovaries twice for five minutes in 0.5%PBST and once for five minutes in 1X PBS. Place a small drop of 1X PBS on to a microscope slide.Use forceps to transfer the abdominal sac from the chambered cover glass to a glass well filled with 1x PBS. Tear apart the abdominal sac and any remaining fat body with forceps. Transfer the ovaries from the well into a drop of 1x PBS on the microscope slide by grasping the center of the ovaries between the tips of the forceps.Add a few drops of 1X PBS to the slide when the solution dries. Once all ovaries have been dissected and transferred to the microscope slide, pipette 40 microliters of mounting medium on to a 22 x 22 millimeter cover slip and place the cover slip gently on top of the ovaries. Finally, let the slide dry overnight prior to imaging.Immunohistochemistry specific to cell proliferation such as phospho histone H3 staining can be used to study cell division patterns of stem cells and other mitotically active cell types. Pupal stalk cells derived from swarm cells can be outlined with Fasciclin III staining. While attempting this procedure, it is best to harvest and dissect more PB than needed for the experiment, give that a few ovaries may become lost during the fat body extraction or immunohistochemistry process.Thanks for watching and good luck with the experiment.

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There are currently over 160 distinct Drosophila cell lines distributed by the Drosophila Genomics Resource Center (DGRC). With genome engineering, the ...

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Dissection, Immunohistochemistry and Mounting of Larval and Adult Drosophila Brains for Optic Lobe Visualization

Dissection, Immunohistochemistry and Mounting of Larval and Adult Drosophila Brains for Optic Lobe Visualization

The Drosophila optic lobe, comprised of four neuropils: the lamina, medulla, lobula and lobula plate, is an excellent model system for exploring the ...