This protocol is an excerpt from Kao&#160;et al., Dissection of&#160;Drosophila melanogaster&#160;Flight Muscles for Omics Approaches,&#160;J. Vis. Exp.&#160;(2019).
1. IFM Dissection After 48 h APF
<ol>
	<li>Assemble necessary equipment including two #5 biology grade forceps, fine scissors, standard glass microscope slides, double-stick tape, pipette, pipette tips, dry ice, and (for RNA applications) isolation reagent (see Table of Materials). Chill the 1x PBS and microcentrifuge tubes on ice.</li>
	<li>Using a lightly wetted paintbrush, transfer the staged pupae to a strip of double-sided sticky tape mounted on a microscope slide (Figure 1A). Place the pupae in a line, oriented in the same orientation (ventral down and anterior towards the bottom of the slide). 
	NOTE: Be careful not to use too much water on the paintbrush or filter, or the pupae will not stick well. If pupae do not stick, dry them by first transferring to a dry filter or tissue paper. Mount as many pupae as can be dissected within a 30 min time window, ideally ~10 pupae.</li>
	<li>Remove the pupa from the pupal case. Use forceps to tease apart and open the pupal case above the anterior spiracles (Figure 1B).</li>
	<li>Gently slide a pair of forceps dorsally towards the posterior, cutting the pupal case as the forceps move (Figure 1B&#39;). Be careful not to rupture the underlying pupa. Liberate the pupa from the opened case and immediately transfer it to a drop of 1x PBS on a second microscope slide (Figure 1B&#34;,C).</li>
	<li>Repeat steps 1.3 and 1.4 for all pupae in the line, then set the double-stick tape slide aside.</li>
	<li>Using the fine scissors, cut the abdomen of the pupa away from the thorax and push it into a separate pile (Figure 1D,D&#39;). Repeat for the remaining pupae. 
	NOTE: Begin timing the length of dissection with step 1.6, as soon as pupal integrity is disrupted. Dissect as many flies as possible in 20&#8211;30 min to prevent cell death and associated transcriptomic and proteomic changes. When dissecting 1 d adults or &#62;90 h pupae, it is often convenient for later steps to additionally remove the head with the fine scissors.</li>
	<li>Using a tissue paper, remove the majority of the 1x PBS (generally cloudy with suspended fat) as well as the pile of abdomens (Figure 1E). Add a drop of fresh, chilled 1x PBS to the remaining thoraxes.</li>
	<li>Use the scissors to cut the thorax in half (Figure 1F,F&#39;) by cutting from the head down the longitudinal body axis in a single motion. Alternately, if the head has been removed, first insert the scissors where the head was attached and cut the top half of the thorax longitudinally between the IFMs. Then, cut the ventral side of the thorax with a second cut in the same orientation.</li>
	<li>Repeat steps 1.7 and 1.8 for all pupae to be dissected, generating a pile of thorax hemisections near the center of the slide. Ensure there is enough chilled 1x PBS on the slide so that the hemisections do not dry out. 
	NOTE: After 48 h APF, IFMs are large enough to be visible under a standard dissecting microscope to the trained eye. At this point in the protocol, muscles with a fluorescent label can be moved to a fluorescent dissecting scope to aid in IFM identification or for training purposes, but this is not necessary.</li>
	<li>Dissect the IFMs out of the thorax. Isolate one of the hemisections using the #5 forceps (Figure 1G,H). Gently insert the tips of one forceps above and below the middle of the IFMs (Figure 1G&#39;,H&#39;). While holding the first forceps still, use fine scissors to cut one end of the IFM away from the cuticle and tendons. Then, cut the other end of the IFM free from the cuticle (Figure 1G&#39;&#39;,H&#39;&#39;). 
	NOTE: Depending on the orientation of the thorax after the first IFM cut, it is useful to rotate the thorax 180&#176; so that the second IFM cut is easier to perform.</li>
	<li>Remove the IFM bundle from the thorax with forceps (Figure 1G&#39;&#39;&#39;,H&#39;&#39;&#39;), transferring it to the edge of the PBS bubble to use water tension to hold it in place (Figure 1I). Push the carcass to the opposite side of the slide. Repeat for the remaining thorax hemisections, generating a collection of dissected IFMs. 
	NOTE: If the IFMs do not stay in a neat pile, remove some of the 1x PBS with a tissue. Be careful not to let all of the PBS evaporate, and ensure that the dissected IFMs and hemithoraxes remain covered by buffer.</li>
	<li>After dissecting all IFMs, quickly perform a quality control on the dissected muscle. Using #5 forceps, remove any jump muscle or cuticle fragments that may have found their way into the sample (Figure 1J-K&#39;&#39;). 
	NOTE: Jump muscle appears different from IFM. If dissecting Mef2-Gal4 labeled muscle under fluorescence, jump muscle has a weaker fluorescence and a different shape and texture. Under normal light, it appears nearly translucent while the IFMs are an opaque, milky yellow (Figure 1J-J&#39;&#39;,K).</li>
	<li>Using water tension, capture (but do not squish) the dissected IFMs between a pair of forceps (Figure 1L). Transfer the IFMs to a 1.5 mL microcentrifuge tube pre-filled with 250 &#956;L of chilled 1x PBS (Figure 1M). Proceed immediately with section 2. 
	NOTE: When forceps tips are brought into proximity of each other and lifted out of a buffer solution, water tension causes a bubble of buffer to be captured between the forceps tips. If IFMs are also present in this bubble, they can be lifted out of the solution and easily transferred to another buffer-filled receptacle. It is important to squeeze the forceps to bring the tips near one another without touching each other, to avoid macerating the tissue captured in the buffer bubble.</li>
</ol>
2. Pellet and Preserve the IFM Sample
<ol>
	<li>Pellet the IFMs by centrifuging the 1.5 mL microcentrifuge tube for 3&#8211;5 min at 2,000 x g in a table-top centrifuge (Figure 2A,B).</li>
	<li>Remove the buffer using a pipette tip (Figure 2C).</li>
	<li>For RNA applications, resuspend the IFM pellet in 50&#8211;100 &#956;L of the desired RNA isolation buffer (see Table of Materials, Figure 2D). Otherwise, proceed to step 2.4. 
	NOTE: IFMs can be dry-frozen after step 2.2 for mass spectrometry preparations or isolation of RNA with commercial kits (see representative results). For RNA applications, better results are obtained by immediately resuspending and freezing the IFM pellet in isolation buffer.</li>
	<li>Freeze sample on dry ice or snap freeze in liquid nitrogen (Figure 2E). Store at -80 &#176;C until ready for subsequent steps in sample preparation for downstream analysis. 
	NOTE: After cryopreservation, samples can be stored for several months before processing for downstream investigation.</li>
</ol>

drosophila late pupa indirect flight muscle (ifm) dissection: a method for high-throughput tissue collection

Source: Kao, S. Y., et al. <a href="https://www.jove.com/video/60309/" target="_blank">Dissection of Drosophila melanogaster Flight Muscles for Omics Approaches</a>. J. Vis. Exp. (2019).
The Drosophila flight muscles are used as a model system for muscle development and physiology. This video describes the flight musculature of adults and highlights a protocol to dissect developing indirect flight muscles that are suitable for RNA sequencing from pupae.

<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/20128/20128fig1.jpg" /> 
Figure 1: Dissection of IFMs after 48 h APF. (A) Aligning of pupae on double-stick tape. (B) Removal of pupae from the pupal case by opening anteriorly, cutting the case dorsally (B&#39;), and lifting out the pupa (B&#39;&#39;). Circle symbols represented the same as Figure 2. (C) Transfer of pupae to buffer. (<stron...

Drosophila Late Pupa Indirect Flight Muscle (IFM) Dissection: A Method for High-Throughput Tissue Collection

Source: Kao, S. Y., et al. Dissection of Drosophila melanogaster Flight Muscles for Omics Approaches. J. Vis. Exp. (2019).
The Drosophila flight muscles ...

- In the adult fly, the indirect flight muscles, or IFMs, are the fly's largest muscles spanning the entire thorax. The IFMs orchestrate the characteristic high frequency wing movements in two-winged flies, including Drosophila. Two opposing muscle fiber groups comprise the IFMs. First, the dorsal longitudinal muscles, or DLMs, are located along the median plane of the thorax above the gut and span from anterior to posterior. Second, the dorsal ventral muscles, or DVMs, span from the top to the bottom of the thorax and are flanked by the TDT, or jump muscle. Both muscle groups are attached to the cuticle. By about 48 hours after puparium formation, or APF, maturing IFMs can be visually identified with a simple dissection microscope. In the example protocol, we will collect IFMs from pupae older than 48 hours APF for high throughput analysis. - For IFM dissection after 48 hours APF, use a lightly wetted paint brush to transfer the staged pupae onto a strip of double-sided sticky tape mounted on a microscope slide. Orient the pupae in a line, ventral side down, and anterior toward the bottom of the slide. When all of the pupae have been placed, use forceps to tease apart and open the first pupal case above the anterior spiracles, and gently slide a pair of forceps dorsally toward the posterior, cutting the pupal case as the forceps move. Free the pupa from the opened case, and immediately transfer the pupa to a drop of PBS on a second microscope slide. When all of the pupae are freed, use fine scissors to cut the abdomen of the pupae away from the thorax and push the abdomens into a separate pile. When all of the thoraxes have been removed, use a piece of tissue paper to remove the majority of the PBS and the pile of abdomens. Add a drop of fresh chilled PBS to the remaining thoraxes before using the scissors to cut from the head along the longitudinal body axis in a single motion to divide the thorax in half. When all of the pupae have been dissected, use the number five forceps to select one of the hemisections and gently insert the tips of one forceps above and below the middle of the IFMs. Holding the first pair of forceps still, use fine scissors to cut one end of the IFM away from the cuticle and tendons before rotating the pupa 180 degrees to allow the other end of the IFM to be cut free from the cuticle and tendons. Use forceps to transfer the IFM bundle from the thorax to the edge of the PBS bubble using water tension to hold the bundle in place. Then, push the carcass to the opposite side of the slide and dissect the rest of the IFMs in the same manner. When all of the IFMs have been collected, use the number five forceps to remove any jump muscle or cuticle fragments that may have found their way into the samples. Then, use water tension to gently capture the dissected IFMs between a pair of forceps and place the IFMs in a 1.5 milliliter microcentrifuge tube containing 250 microliters of chilled PBS.

drosophila-late-pupa-indirect-flight-muscle-ifm-dissection-method-for

Research

JoVE Encyclopedia of Experiments

Biology

Drosophila melanogaster (fruit fly)

Pupa

The Drosophila flight muscles are used as a model system for muscle development and physiology. This video describes the flight musculature of adults and highlights a protocol to dissect developing indirect flight muscles that are suitable for RNA sequencing from pupae.

Overview

Protocol

<img title="figure-representative results-25" alt="figure-representative results-25" class="xfigimg" src="/files/ftp_upload/20128/20128fig1.jpg" /> 
Figure 1: Dissection of IFMs after 48 h APF. (A) Aligning of pupae on double-stick tape. (B) Removal of pupae from the pupal case by opening anteriorly, cutting the case dorsally (B&#39;), and lifting out the pupa (B&#39;&#39;). Circle symbols represented the same as Figure 2. (C) Transfer of pupae to buffer. (<stron...

Drosophila Late Pupa Indirect Flight Muscle (IFM) Dissection: A Method for High-Throughput Tissue Collection

In This Article

Overview

Protocol

Representative Results