This protocol is an excerpt from&#160;Guan&#160;et al., Visualize&#160;Drosophila&#160;Leg Motor Neuron Axons Through the Adult Cuticle,&#160;J. Vis. Exp.&#160;(2018).
1. Leg Dissection and Fixation
<ol>
	<li>Take a glass multi-well plate and fill appropriate number of wells with 70% ethanol. Add 15&#8211;20 CO2-anesthetized flies (of either sex and any age) to each well and by using a brush, gently dab the flies into the ethanol solution until flies are fully submerged. 
	NOTE: This step is to remove the hydrophobicity of the cuticle. Do not wash for more than 1 min, because this increases auto-fluorescence of the cuticle.</li>
	<li>Rinse the flies 3 times with 0.3% nonionic surfactant detergent solution in 1x phosphate buffered saline (PBS). Keep the flies in this solution for at least 10 min. 
	NOTE: Legs are better fixed when including detergent, which is likely to increase penetration of the fixative inside the leg.</li>
	<li>Place a multi-well plate on the ice along with a tube of 4% paraformaldehyde (PFA). 
	NOTE: The preparation of fresh 4% PFA from a 16% PFA ethanol free stock solution is critical.</li>
	<li>Use forceps to remove the head and abdomen of the flies without damaging the thoracic segment or the legs. 
	NOTE: Removing the abdomen makes it easier to hold the fly and dissect the legs.</li>
	<li>Dissect the legs from the thoracic segment with forceps and place the legs in the wells containing 4% PFA. For this, push gently but firmly at the coxa-thorax junction using the tip of fine forceps until the leg detaches.</li>
	<li>Fix the legs in 4% PFA overnight at 4 &#176;C (approximately 20 h total).</li>
	<li>Wash the legs with 0.3% nonionic surfactant detergent solution in 1x PBS, 5x for 20 min each.</li>
	<li>Replace the washing buffer with mounting medium. Keep the leg in mounting medium for at least a day prior to mounting to allow for its complete penetration into the leg. 
	NOTE: If the mounting medium is highly viscous, dilute the mounting medium to 80% with 1x PBS because the sudden change in viscosity can cause the cuticle to collapse and damage the overall structure of the leg. Depending on the Gal4 driver, flies can be preserved for 1 to 3 weeks at 4 &#176;C in mounting media until mounting.</li>
</ol>
2. Leg Mounting
<ol>
	<li>Place approximately 20 &#181;L of 70% glycerol on the left-hand side of a microscope slide and cover with a square 22 x 22 mm2 coverslip (Figure 1A,B). Pipette about 10 &#181;L of mounting medium along a line parallel to and at a small distance from the right edge of this coverslip. Add another 30 &#181;L of mounting medium further on the right side of the slide (Figure 1C). 
	NOTE: When applying a coverslip over the legs (see below) the mounting medium will gently spread under the coverslip and around the legs without displacing them.</li>
	<li>Using fine forceps lift the leg from the solution and gently put it on the mounting medium near the left coverslip. Do the same for each leg and align them from top to bottom (Figure 1D).
	<ol>
		<li>Lift and transfer the legs in a drop of medium held between both tips of the forceps. Orient the legs in two ways: external side up or down.</li>
	</ol>
	</li>
	<li>Once all the legs (up to 6&#8211;8 legs can be mounted) are properly aligned, put a second coverslip over the legs such that this coverslip rests slightly on the previously placed coverslip Figure 1E to allow for space between the coverslip and the tissue and to prevent the legs from getting damaged (Figure 1G). 
	NOTE: Alternatively, use sticker wells or orthodontic wax to create space between the coverslip and the slide.</li>
	<li>Use a nail polish to secure the position of the coverslips at each corner (Figure 1F). 
	NOTE: The tissue is now ready to image.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Ethanol absolute</td>
			<td>Fisher</td>
			<td>E/6550DF/17</td>
			<td>Absolute analytical reagent grade</td>
		</tr>
		<tr>
			<td>nonionic surfactant detergent</td>
			<td>Sigma-Aldrich</td>
			<td>T8787</td>
			<td>Triton X-100, for molecular biology</td>
		</tr>
		<tr>
			<td>Fine forceps</td>
			<td>Sigma-Aldrich</td>
			<td>F6521</td>
			<td>Jewelers forceps, Dumont No. 5</td>
		</tr>
		<tr>
			<td>Glass multi-well plate</td>
			<td>Electron Microscopy Sciences</td>
			<td>71563-01</td>
			<td>9 cavity Pyrex, 100 mm x 85 mm</td>
		</tr>
		<tr>
			<td>PFA</td>
			<td>Thermofisher</td>
			<td>28908</td>
			<td>Pierc 16% Formaldehyde (w/v), Methanol-free</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Fisher BioReagents</td>
			<td>BP 229-1</td>
			<td>Glycerol (Molecular Biology)</td>
		</tr>
		<tr>
			<td>Spacers</td>
			<td>Sun Jin Lab Co</td>
			<td>IS006</td>
			<td>iSpacer, four wells, around 12 &#956;L working volume per well, 7 mm diameter, 0.18 mm deep</td>
		</tr>
		<tr>
			<td>Square 22 mm x 22 mm coverslips</td>
			<td>Fisher Scientific</td>
			<td>FIS#12-541-B</td>
			<td>No. 1.5-0.16 to 0.19 mm thick</td>
		</tr>
		<tr>
			<td>Mounting Medium</td>
			<td>Vector Laboratories</td>
			<td>H-1000</td>
			<td>Vectashield Antifade Mounting Medium</td>
		</tr>
	</tbody>
</table>

processing insect legs for fluorescence microscopy: a method to preserve neuromuscular structures for imaging

Source: Guan, W., et al. <a href="https://www.jove.com/video/58365/" target="_blank">Visualize Drosophila Leg Motor Neuron Axons Through the Adult Cuticle</a>. J. Vis. Exp. (2018).
This video describes a method to dissect, fix, and mount the Drosophila adult leg while preserving its neuromusculature intact for imaging analysis.

<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/20121/20121fig1.jpg" /> 
Figure 1: Procedure to mount legs on microscope slides. <a href="https://www.jove.com/files/ftp_upload/20121/20121fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Processing Insect Legs for Fluorescence Microscopy: A Method to Preserve Neuromuscular Structures for Imaging

Source: Guan, W., et al. Visualize Drosophila Leg Motor Neuron Axons Through the Adult Cuticle. J. Vis. Exp. (2018).
This video describes a method to ...

- To begin, submerge anesthetized insects in 70% ethanol solution to reduce the hydrophobicity of the cuticle. Then, wash and permeabilize the tissue with phosphate-buffered saline containing a detergent for at least 30 minutes to allow tissue penetration during fixation. Next, remove the head and abdomen from the thorax. Use forceps to apply gentle pressure to the coxa-thorax junction, and detach the leg. Carefully place the legs in ice-cold 4% paraformaldehyde solution overnight to allow it to penetrate the tissue. Paraformaldehyde fixes tissues by cross-linking proteins. Then, remove the fixation solution and wash the tissue several times with phosphate-buffered saline containing detergent. Before mounting, place the legs in pure or slightly diluted mounting buffer for at least one day to provide enough time for the buffer to enter the tissue. Lastly, mount the legs in a drop of mounting medium. Use a spacer between the microscope slide and the coverslip to prevent the specimen from damage, and secure the mount with nail polish. In the following example, we will see the dissection, fixation, and mounting of Drosophila melanogaster legs. - Begin by filling the appropriate number of wells in a glass multiwell plate with 70% ethanol. Use a brush to add 15 to 20 carbon dioxide anesthetized flies of any age or sex to each well, and gently dab the flies into the ethanol until they are fully submerged. After no more than one minute, rinse the flies three times with 0.3% nonionic surfactant detergent solution in phosphate-buffered saline for at least 10 minutes per wash. After the last wash, use forceps to remove the head and abdomen of each fly without damaging the thoracic segment or the legs, and use the tip of a pair of fine forceps to gently but firmly apply pressure to the coxa-thorax junction to detach one leg from the thoracic segment. Place the legs in one well of a new multiwell plate, containing freshly prepared 4% paraformaldehyde on ice, for an overnight incubation at 4 degrees Celsius. - It is important to push the legs gently into the fixation buffer without letting them float to obtain well-fixed legs. - The next day, wash the legs five times in fresh 0.3% nonionic surfactant detergent solution for 20 minutes per wash. After the last wash, replace the detergent with mounting medium and keep the legs in the mounting medium for at least 24 hours. The next day, add approximately 20 microliters of 70% glycerol next to the coated end of the glass microscope slide and cover the glycerol with a 22 by 22 millimeter coverslip. Next, add in about 10-microliter line of mounting medium to the right of the coverslip and apply a second 30-microliter line of mounting medium to the right of the 10-microliter line. Using fine forceps, transfer one leg from the multiwell plate in a drop of medium to the 10-microliter strip of mounting medium in an external side up or down orientation. Repeat until six to eight legs have been mounted and aligned, and place a second coverslip over the legs such that the second coverslip rests slightly on the first coverslip. Then, use nail polish at each corner of the coverslips to secure them in place.

processing-insect-legs-for-fluorescence-microscopy-method-to-preserve

Research

JoVE Encyclopedia of Experiments

Biology

Drosophila melanogaster (fruit fly)

Adult

This video describes a method to dissect, fix, and mount the Drosophila adult leg while preserving its neuromusculature intact for imaging analysis.

Overview

Protocol

<img title="figure-results-25" alt="figure-results-25" class="xfigimg" src="/files/ftp_upload/20121/20121fig1.jpg" /> 
Figure 1: Procedure to mount legs on microscope slides. <a href="https://www.jove.com/files/ftp_upload/20121/20121fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Processing Insect Legs for Fluorescence Microscopy: A Method to Preserve Neuromuscular Structures for Imaging

In This Article

Overview

Protocol

Results

Materials

Name	Company	Catalog Number	Comments
Ethanol absolute	Fisher	E/6550DF/17	Absolute analytical reagent grade
nonionic surfactant detergent	Sigma-Aldrich	T8787	Triton X-100, for molecular biology
Fine forceps	Sigma-Aldrich	F6521	Jewelers forceps, Dumont No. 5
Glass multi-well plate	Electron Microscopy Sciences	71563-01	9 cavity Pyrex, 100 mm x 85 mm
PFA	Thermofisher	28908	Pierc 16% Formaldehyde (w/v), Methanol-free
Glycerol	Fisher BioReagents	BP 229-1	Glycerol (Molecular Biology)
Spacers	Sun Jin Lab Co	IS006	iSpacer, four wells, around 12 μL working volume per well, 7 mm diameter, 0.18 mm deep
Square 22 mm x 22 mm coverslips	Fisher Scientific	FIS#12-541-B	No. 1.5-0.16 to 0.19 mm thick
Mounting Medium	Vector Laboratories	H-1000	Vectashield Antifade Mounting Medium