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 ...

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