Small Scale Dissection and Antibody Staining of Eye-Antennal and Wing Imaginal Discs from Drosophila melanogaster

Summary

This article describes an efficient imaginal disc dissection and antibody staining protocol that is economical with reagents and facilitates handling of imaginal discs in small numbers.

Abstract

Drosophila imaginal discs have long been studied as models of epithelial development, patterning, and regeneration. Antibody labeling and imaging of dissected imaginal discs is important for visualizing protein expression patterns as markers of developmental and physiological processes. This method of imaginal disc dissection and labeling was originally developed for immune-electron microscopy of eye-antennal imaginal discs by Tomlinson and Ready. Using 60-microwell plates, and by transferring tissues between solutions rather than transferring solutions, individualized attention to tissue samples is possible with very little wastage. Even single imaginal discs can be processed. The method requires only small volumes of antibodies and other reagents. As an example of the method’s application to other small tissues, the article also demonstrates how wing imaginal discs can be dissected and labeled by the same method. A similar approach can be applied to any small tissue of similar dimensions, and also to other histochemical and labeling procedures besides antibody staining.

Introduction

Drosophila imaginal discs are progenitor tissues that are set aside during embryogenesis and grow within the larva without differentiating and contribute to the adult epidermal structures during the pupal stage¹. Their study has provided many insights into developmental patterning mechanisms, with the eye and wing imaginal discs having been particularly well studied^2,3,4,5,6. Immunohistochemistry is particularly useful to investigate spatial and temporal patterns of gene exp....

Protocol

The reagents and the equipment used are listed in the Table of Materials.

1. Making the transfer tool

1. Use pliers to cut ~2.5cm of 0.005” diameter tungsten wire. Use forceps to form one end of the wire into a hook or a loop (Figure 3). Insert the other end into a needle holder. This will be used to transfer the discs with little or no liquid transfer. The loop may be the easier of the two to use, but the hook transfers less liquid between solutions.

2. Dissecting and fixing eye-antennal imaginal discs

....

Results

The Tomlinson and Ready procedure regularly yields excellent antibody staining results. Here, triply labeled eye, antennal, and wing imaginal discs are illustrated. In Figure 5A, a third-instar eye-antennal imaginal disc has been triple-labeled to detect the transcription factor Senseless (blue), neural progenitor marker Elav (red), and anti-GFP labeling of cell clones expressing RNAi for the Ocho transcript (green). In Figure 5B, a third instar wing im.......

Discussion

Antibody staining of Drosophila tissues using 60-well conical-well plates using the Tomlinson and Ready method results in excellent results and is highly reliable, as each imaginal disc is followed under the dissecting microscope during each manipulation. The method requires only small amounts of antibody reagents, and exchanges solutions more completely with each step since samples are transferred with very little associated liquid. It is particularly suited to handling modest numbers of samples that may easily.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Disodium Phosphate	Fisher	7558-79-4	Na2HPO4 - sodium phosphate dibasic anhydrous. Other hydration states can be used but change the molecular weight
Forceps: Dumont #5 Biologie forceps	Fine science tools	11252-20	Style: #5
			Tip Shape: Straight
			Length: 11cm
			Autoclave Safe: Yes
			Tips: Biology
			Tip Dimensions:
			0.05 x 0.02mm
			Alloy / Material: Inox
Formaldehyde	Fisher	BP531-500	Molecular Biology-grade, 37% Formaldehyde. 500ml
Formaldehyde Fixative	Fisher	BP531-500	3.7% formaldehyde in 0.1M Sodium Phosphate Buffer pH7.2 made by dilution from 37% stock.
Glycerol	Fisher Bioreagents	BP229-1
Ice	User Preference
L-Lysine, 98%	Thermoscientific	J62225.22
mAb40-1	DHSB	40-1a-s	Anti-beta galactosidase
Microcentrifuge tubes	USA scientific	1615-5520	1.5mL tubes to hold samples.
Microdissection Scissors	Fine Science Tools	15003-08	5mm cutting edge, straight tip, 8.5cm
Monosodium Phosphate	Fisher	7558-80-7	NaH2PO4 - Sodium Phosphate Monobasic anhydrous. If using a hydrated form, the weight needed to make a 0.1M solution would change.
Mounting Medium			75% glycerol solution in 0.1M sodium phosphate buffer pH7.2 with 2.5% n-propyl gallate. Mix by inversion to minimize bubbles.
NSG			0.1M sodium phosphate buffer pH7.2, 0.1% saponin, 5% NGS
n-propyl gallate	MP Biomedicals	210274780
Nail Polish	Ted Pella, Purchased from Fisher	NC0381432	For sealing slides
Needle holder	Fisher	NC0099380	For microdissecting needles. Hollow stainless steel; handle length: 4 3/4" long
Nunc Microwell MiniTrays	Thermo Scientific	12565155	MicroWell MiniTray for serological applications. Well count 60
Parafilm	StatLab	PM996	StatLab ParaFilm M Self-Sealing Flexible Film
Paraformaldehyde	Polysciences, Inc.	00380-250
10x PBS	User Preference		Prepare 800 mL of distilled water in a suitable container.
			Add 1.37M of Sodium chloride to the solution.
			Add 26.8mM of Potassium Chloride to the solution.
			Add 101mM of Sodium Phosphate Dibasic to the solution.
			Add 18.0mM of Potassium Phosphate Monobasic to the solution.
			Adjust solution to desired pH (typically pH ≈ 7.4).
			Add distilled water until the volume is 1 L.
PBT	User Preference		0.1M sodium phosphate buffer pH7.2, 0.1% bovine serum albumin, and 0.2% Triton X-100.
PDT	User Preference		0.1M Sodium Phosphate buffer (pH = 7.2), 0.3% deoxycholate, and 0.3% Triton x-100
Petri Dish	Falcon	Corning 351007	Petri Dish; 60mm; non treated
Petri Dish	Falcon	Corning 351008	Petri Dish; 35mm; non treated
Pliers	User Preference
PLP	n/a		2% paraformaldehyde 1.35% lysine in 0.05M sodium phosphate buffer pH7.2
Secondary Antibodies	Jackson ImmunoResearch	715-545-151	0.5mg Alexa Fluor 488 Donkey Anti-Mouse IgG.
Slide Covers	VWR	48393-172	VWR Coverglass #1.5 22x40 (CASE)
Slides	Fisher	125442	Fisher Glass Slides 10bx/CS
Sodium Deoxycholate	Thermo Fisher Sci	89904	Sodium Deoxycholate Detergent; 5mg
Sodium Periodate	Sigma Aldrich	71859-25G	Sodium Periodate – 25 grams
Sodium Phosphate Buffer			0.1M NaPO4 buffer pH 7.2. Mix 28ml of 0.1M anyhdrous monosodium phosphate stock (11.998g NaH2PO4 to 1L of distilled water) with 72ml of 0.1M anyhydrous disodium phosphate stock (14.196g Na2HPO4 to 1L of distilled water) to generate 100ml of 0.1M NaPO4 buffer. Alternatively, PBS may be used.
Tungsten wire	Ted Pella	27-11	.005” dia.
Triton x-100	Thermo Fisher Sci	A16046AE
Vortex	User Preference