Dissection and Immunostaining of Imaginal Discs from Drosophila melanogaster

Summary

The adult structures of Drosophila are derived from sac-like structures called imaginal discs. Analysis of these discs provides insight into many developmental processes including tissue determination, compartment boundary establishment, cell proliferation, cell fate specification, and planar cell polarity. This protocol is used to prepare imaginal discs for light/fluorescent microscopy.

Abstract

A significant portion of post-embryonic development in the fruit fly, Drosophila melanogaster, takes place within a set of sac-like structures called imaginal discs. These discs give rise to a high percentage of adult structures that are found within the adult fly. Here we describe a protocol that has been optimized to recover these discs and prepare them for analysis with antibodies, transcriptional reporters and protein traps. This procedure is best suited for thin tissues like imaginal discs, but can be easily modified for use with thicker tissues such as the larval brain and adult ovary. The written protocol and accompanying video will guide the reader/viewer through the dissection of third instar larvae, fixation of tissue, and treatment of imaginal discs with antibodies. The protocol can be used to dissect imaginal discs from younger first and second instar larvae as well. The advantage of this protocol is that it is relatively short and it has been optimized for the high quality preservation of the dissected tissue. Another advantage is that the fixation procedure that is employed works well with the overwhelming number of antibodies that recognize Drosophila proteins. In our experience, there is a very small number of sensitive antibodies that do not work well with this procedure. In these situations, the remedy appears to be to use an alternate fixation cocktail while continuing to follow the guidelines that we have set forth for the dissection steps and antibody incubations.

Introduction

For more than a century the fruit fly, Drosophila melanogaster, has been a premier system to study development, behavior and physiology. Development in the fly can be divided into two broad stages: embryonic and post-embryonic with much of the latter taking place within monolayer epithelia called imaginal discs ^1-3. Drawings of imaginal discs were first published in 1864 by August Weismann as part of his broad monograph on insect development ¹. These discs begin their development during embryogenesis, are patterned during the larval stages, survive the massive histolysis of the early pupal stages, and ultimately give ris....

Protocol

1. Preparation of Larvae

1. Fill a 35 mm Petri dish with dissection buffer.
2. Place larvae in Petri dish and allow them to swim around for a few minutes (self-cleaning step).
3. Transfer larvae to a pool of dissection buffer on a silicone-based dissection plate. This pool should be at one edge of the plate. The dissection plate consists of a silicone solution that has been poured and hardened within a glass Petri dish.
4. Using a pasteur-pipet, place a larger pool of dissection buffer in the middle of the dissection plate.
5. Using #5 forceps, transfer a single cleaned larva from the small pool to the larger pool of dissection buf....

Results

The method that is described above reliably produces high quality material for analysis with in situ probes, transcriptional reporters, protein traps and antibodies. In Figure 1 we display eye-antenna, genital, wing, haltere and leg discs that are routinely recovered with this method. These discs have been treated with a phalloidin-conjugated fluorophore, which binds to F-actin and therefore outlines each cell. If the tissue has been fixed properly then the morphogenetic furrow of the eye disc, .......

Discussion

Although this procedure has largely focused on the isolation and subsequent treatment of eye-antennal discs, it is amenable to being used to isolate and analyze the wing, haltere, leg and genital discs (Figure 4). The only required modification of the protocol for isolating these discs (as opposed to the eye-antennal disc) is the method of coarse dissection (section 2 of the protocol). The first thoracic leg (T1) pair is found at the anterior of the larva and can be recovered by following the protoc.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Sylgard 184 Silicone Elastomer Kit	Dow Corning	184 SIL ELAST KIT 0.5 KG	Used to create base for dissection plate
Pryex Glass Petri Dish 150 x 20 mm	Dow Corning	3160-152CO	Use the cover for dissection plate
#5 Dissecting Forceps	Ted Pella	525	Forceps must be kept very sharp
9 well watch glass	Vairous Vendors		Used for fixation of imaginal disc complexes
50 ml Erlenmeyer Flask	Various Vendors
Small Stir Bar	Various Vendors		Small enough to fit into Erlenmeyer Flask
50 ml Conical Tubes	Various Vendors
1.5 ml Microfuge Tubes	Various Vendors		Clear or Dark depending upon application
Microfuge Rack	Various Vendors
Benchtop Rotator	Various Vendors		100 μl volume should not splatter at low setting
Paraformaldehyde	Macron Chemicals	2-26555-1	Serves as fixative
Sodium Phosphate Monobasic	Sigma Chemical Co	S-3139	Used to make dissection and wash buffers
Sodium Phosphate Dibasic	Sigma Chemical Co	71636	Used to make dissection and wash buffers
Lysine	Acros Organics	125221000	Used in the fixative solution
Sodium Periodate	Sigma Chemical Co	S-1878	Used in the fixative solution
Triton X-100	EMD Chemicals	MTX1568-1	Used to perforate imaginal discs
Sodium Hydroxide	EM Science	SX0593-3	Used to dissolve paraformaldehyde
100% Normal Goat Serum	Jackson Laboratories	005-000-121	Serves as a blocking solution
Primary Antibodies	Various Vendors		Dilute in 10% goat serum as directed by manufacturer
Secondary Antibodies	Various Vendors		Dilute in 10% goat serum as directed by manufacturer
Vectashield	Molecular Probes	H-1000	Prevents bleaching of samples
Microscope Slides	Fischer Scientific	48312-003
Glass Cover Slips 18 x 18 mm	Fischer Scientific	12-542A
Kimwipe Tissue	Various Vendors		Prevents Glass slides from adhering to silicone base
Panit Brush 000	Various Vendors		Use to gently lower coverslip on to samples