Removal of Drosophila Muscle Tissue from Larval Fillets for Immunofluorescence Analysis of Sensory Neurons and Epidermal Cells

Summary

Studies of neuronal morphogenesis using Drosophila larval dendritic arborization (da) neurons benefit from in situ visualization of neuronal and epidermal proteins by immunofluorescence. We describe a procedure that improves immunofluorescence analysis of da neurons and surrounding epidermal cells by removing muscle tissue from the larval body wall.

Abstract

Drosophila larval dendritic arborization (da) neurons are a popular model for investigating mechanisms of neuronal morphogenesis. Da neurons develop in communication with the epidermal cells they innervate and thus their analysis benefits from in situ visualization of both neuronally and epidermally expressed proteins by immunofluorescence. Traditional methods of preparing larval fillets for immunofluorescence experiments leave intact the muscle tissue that covers most of the body wall, presenting several challenges to imaging neuronal and epidermal proteins. Here we describe a method for removing muscle tissue from Drosophila larval fillets. This protocol enables imaging of proteins that are otherwise obscured by muscle tissue, improves signal to noise ratio, and facilitates the use of super-resolution microscopy to study da neuron development.

Introduction

Drosophila larval dendritic arborization (da) neurons provide a valuable model for studying neuronal development due to their amenability to genetic manipulation and the ease with which they can be imaged. These sensory neurons have been instrumental in the identification of numerous pathways that control dendrite morphogenesis^1-3.

Four classes of da neurons (class I - IV) innervate the larval epidermis. These neurons lie between the basement membrane and the epidermis, with their dendrites forming largely two-dimensional arrays^4,5. Of the four classes, class IV da neurons have the most highly branched arbors and, like sensory neurons of other animals, the elaboration of these arbors requires intrinsic factors as well as cues from neighboring tissues, particularly the epidermis, for their development^6-9.

Studies to determine how such neuronal and extra-neuronal factors control dendrite morphogenesis benefit from the ability to detect protein expression in situ by immunofluorescence. The outer cuticle of the larva is impenetrable to antibodies, but this impediment is easily overcome by the preparation of larval fillets through well-established dissection methods^10,11. However, the body wall muscle tissue that lies just interior to the basement membrane presents several challenges towards visualization of da neurons and epidermal cells. First, the muscle tissue, which lines most of the body wall, greatly obscures fluorescent signals emanating from neuronal or epidermal tissue. This substantially reduces the signal to noise ratio in the sample. Second, many relevant proteins may be expressed in the muscle tissue as well as in the neurons or epidermis. This muscle-derived fluorescence signal is likely to further obscure detection of a fluorescence signal from the neuron or epidermis. Finally, advances in microscopy technologies now permit imaging of samples at sub-diffraction resolution and may be especially helpful in discerning the localization of proteins that are expressed in neurons and surrounding epidermal cells^12,13. However, imaging via super-resolution microscopy benefits from a strong signal to noise ratio and close proximity of the sample to the coverslip. In addition to reducing the signal to noise ratio, the larval body wall muscle distances da neurons from the coverslip, thereby limiting the improved image resolution that can be achieved with super-resolution microscopy methods. Besides challenges for immunofluorescence analysis, muscle tissue presents a barrier to electrophysiological recording from sensory neurons in the larval body wall. Its removal therefore benefits neurophysiological manipulation of sensory neurons¹⁴.

Here a method for manual removal of Drosophila larval muscle tissue is described. We demonstrate that our protocol permits immunofluorescence imaging of proteins that are otherwise obscured by muscle tissue, improves the signal to noise ratio for visualization of class IV da neurons, and enables the use of super-resolution microscopy to better discriminate spatial relationships of proteins and cellular structures in da neurons and the epidermis.

Protocol

Note: The procedure for muscle removal (Figure 1) is a modification of previously described methods for preparing larval fillets. The steps that precede and follow muscle removal are outlined briefly and the reader is referred to previous work ^{10, 11} for more detailed descriptions.

1. Dissect Larva in Cold Saline

1. Prepare a working dilution of cold HL3.1 saline¹⁵ or cold Ca²⁺-free HL3.1 saline¹¹ (Table 1). Place the larva in a silicone elastomer dish with just enough cold saline to cover the bottom of the dish.
   NOTE: See Discussion regarding the choice of saline.

1x HL3.1 saline (pH 7.2)

5 mM HEPES

70 mM NaCl

5 mM KCl

1.5 mM CaCl2   (omit for Ca2+-free saline)

4 mM MgCl2

10 mM NaHCO3

5 mM trehalose

115 mM sucrose

Filter sterilize and store at 4 °C

Note: Composition mimics insect hemolymph

Table 1. Composition of Cold Saline.

2. Position the larva with the ventral side up. The ventral surface of the larva can be identified by the abdominal dentical belts and the dorsal side by the primary larval tracheal tubes running from anterior to posterior. Stretch the larva in the anterior-posterior direction and pin the head and tail to a silicone elastomer dissecting dish using insect pins. Use fine dissecting scissors to cut along the ventral midline, beginning at one end and progressing toward the other.
   NOTE: This orientation preserves the commonly studied dorsal cluster of da neurons.
3. After the larva is cut open, pin the four corners to the dissecting dish as though opening a book. Use forceps to grab and remove internal organs including the CNS, gut, and trachea. Adjust insect pins so that the fillet is taut but not maximally stretched.
   NOTE: Muscles are anchored to the body wall at segmental boundaries. Although muscles cover most of the body wall, they are absent in a narrow region near the dorsal midline.

2. Muscle Removal

1. Locate the dorsal midline of the larva, where muscle tissue is absent. Position a single forceps prong such that it can be inserted underneath the muscle tissue in the flattest possible orientation.
2. Starting at the dorsal midline, near the anterior boundary of the segment, carefully slide the forceps prong between the muscle and epidermis, taking care to minimize contact between the forceps and epidermis.
3. Pull the forceps upwards to break the attachment of the muscle to the body wall at one anchor point. Repeat this process for the remaining hemi-segment(s) of interest.
   NOTE: This protocol optimizes preservation of the posterior of each da neuron dendrite field. To preserve the anterior part of the dendrite field, it is best to insert the forceps prong at the posterior end of each segment.
4. Re-adjust insect pins such that the larval fillet is maximally stretched in all directions.
5. Fix the fillet while it is still pinned to the dissecting dish using cold, freshly prepared 4% formaldehyde in PBS for 25 min.
6. Rinse 5 times in PBS.
7. Use forceps to carefully pull away muscle tissue from the remaining anchor points, taking care to minimize contact with the epidermis.
8. Unpin and remove the fillet from the dissecting dish to a 1.5 ml microcentrifuge tube. Perform all subsequent washing, blocking, and immunofluorescence staining steps, as previously described¹¹.

3. Mount the Larval Fillet

1. First remove the head and tail using fine dissecting scissors in order to make the sample as flat as possible. Mount the fillet on a coverslip in antifade mountant with the inner surface of the fillet facing the coverslip.
2. Place a microscope slide on the coverslip and press gently to disperse the mounting medium. Flip the microscope slide over and seal the coverslip on the slide using nail polish. Slides can be stored at -20 °C for at least one month.

Results

We demonstrate the utility of the muscle removal procedure for improving signal to noise ratio in immunofluorescence experiments to co-visualize the septate junction proteins Coracle (Cora) and Discs-large (Dlg) together with class IV da neurons labeled with the membrane marker CD4-tdTomato.

Cora has been previously used to identify tracts where da neuron dendrites are enclosed by epidermal cells and is one of many identified epidermal factors that have been st...

Discussion

Here a protocol is described for manual removal of muscle tissue from Drosophila larval fillets. This protocol modifies previously described larval dissection techniques^10,11. After the larva is dissected in a silicone elastomer dish, the dorsal midline is located. A single forceps prong, in its flattest possible orientation, is carefully inserted between the muscle tissue and the epidermis, near the dorsal midline. The forceps are gently pulled upwards to separate muscle tissue from one anchor point ...

Materials

Name	Company	Catalog Number	Comments
Dumont #5 tweezers	Electron Microscopy Sciences	72701-D
Micro Scissors, 8 cm, straight, 5 mm blades, 0.1 mm tips	World Precision Instruments	14003
Sylgard 184 silicone elastomer kit	Dow Corning	3097358-1004	for dissecting plates
Austerlitz insect pins, 0.1 mm	Fine Science Tools	26002-10
Fostec 8375 light source	Artisan Technology Group	62792-4
Zeiss Stemi 2000	Carl Zeiss Microscopy
Vectashield antifade mounting medium	Vector Laboratories	H-1000	for confocal microscopy
Prolong Diamond antifade mountant	Life Technologies	P36970	for structured illumination microscopy
Micro cover glass, 22 x 22 mm, No. 1.5	VWR	48366-227
Superfrost Plus microscope slides, 25 x 75 x 1.0 mm	Fisherbrand	12-550-15
Mouse anti-Coracle antibody	Developmental Studies Hybridoma Bank	C615.16	supernatant, dilute 1:50
Mouse anti-Discs large antibody	Developmental Studies Hybridoma Bank	4F3	supernatant, dilute 1:50
Rabbit anti-dsRed antibody	Clontech	632496	dilute 1:1,000
Goat anti-rabbit antibody, Alexa Fluor 568 conjugated	ThermoFisher Scientific	A-11011	dilute 1:1,000
Goat anti-mouse antibody, Alexa Fluor 488 conjugated	ThermoFisher Scientific	A-11001	dilute 1:500