Dissection of the Drosophila Pupal Retina for Immunohistochemistry, Western Analysis, and RNA Isolation

Summary

This paper presents a surgical method for dissecting Drosophila pupal retinas along with protocols for the processing of tissue for immunohistochemistry, western analysis, and RNA-extraction. 

Abstract

The Drosophila pupal retina provides an excellent model system for the study of morphogenetic processes during development. In this paper, we present a reliable protocol for the dissection of the delicate Drosophila pupal retina. Our surgical approach utilizes readily-available microdissection tools to open pupae and precisely extract eye-brain complexes. These can be fixed, subjected to immunohistochemistry, and retinas then mounted onto microscope slides and imaged if the goal is to detect cellular or subcellular structures. Alternatively, unfixed retinas can be isolated from brain tissue, lysed in appropriate buffers and utilized for protein gel electrophoresis or mRNA extraction (to assess protein or gene expression, respectively). Significant practice and patience may be required to master the microdissection protocol described, but once mastered, the protocol enables relatively quick isolation of mainly undamaged retinas.

Introduction

The Drosophila retina is composed of approximately 750 ommatidia surrounded by pigment cells arranged in a honeycomb lattice^1,2,3,4. Each ommatidium contains eight photoreceptor neurons, four lens-secreting cone cells, and two primary pigment cells. Surrounding each ommatidium are pigment-producing lattice cells and sensory bristle groups. Due to its post-mitotic nature and stereotypical hexagonal arrangement, the Drosophila pupal retina provides an excellent model system for the study of morphogenetic processes including c....

Protocol

1. Tissue Preparation

1. Set up Drosophila crosses (as described previously²⁰) or culture specific Drosophila strains to obtain pupae of the desired genotype. To ensure that a large number of pupae emerge coincidently, establish these fly cultures in duplicate on nutrient-rich food media or standard food media generously supplemented with yeast-paste.
2. Maintain Drosophila cultures at 25 °C. For crosses utilizing the UAS-GAL4 system, GMR-GAL4

Discussion

The method of Drosophila pupal eye dissection described here allows for the isolation of 6 to 10 eye-brain complexes within 10 to 15 min. However, patience and practice are essential in order to master the dissection technique and improve the quality and speed of dissections. This short dissection time ensures that each eye is approximately the same developmental stage, reducing variability in the phenotype or gene expression of retinas in a data set. Whilst alternative protocols may require less practice to mas.......

Materials

Name	Company	Catalog Number	Comments
Adobe Photoshop	Adobe		Image processing software
Bamboo splints, 6"	Ted Pella Inc	116
Beta mercaptoethanol	Sigma-Aldrich	M3148
Beta-glycerol phosphate	Sigma-Aldrich	50020
Black dissecting dish			Glass petri dish filled to rim with SYLG170 or SYLG184 (colored black with finely ground charcoal powder). Leave at room temperature for 24-48 h to polymerize.
Blade holder	Fine Science Tools	10053
Bovine serum albumin	Sigma-Aldrich	A7906
cOmplete, EDTA-free protease inhibitor cocktail tablets	Roche	4693132001
Confocal microscope (Zeiss LSM 501)	Carl Zeiss		or similar microscope
Diethyl Pyrocarbonate (DEPC)	Sigma-Aldrich	40718
Double-sided tape	3M	665
Drosophila food media, nutrient-rich			7.5% sucrose, 15% glucose, 2.5% agar, 20% brewers yeast, 5% peptone, 0.125% MgSO4.7H2O, 0.125% CaCl2.2H20
Drosophila food media, standard			Bloomington Drosophila Stock center cornmeal recipe.  (https://bdsc.indiana.edu/information/recipes/bloomfood.html)
Ethylenediaminetetraacetic acid	Sigma-Aldrich	E6758
Fixative solution			4% formadehyde in PBS, pH 7.4.
Fluorescence microscope (TCS SP5 DM microscope)	Leica Microsystems		or similar microscope
Forceps	Fine Science Tools	91150-20	Forceps should be sharpened frequently.
Formaldehyde	Thermo Scientific	28908
Glass 9-well dishes	Corning	7220-85	Also known as 9-well dishes
Glass coverslips (22 x 22 mm)	Fisher Scientific	12-542-B
Glass microscope slides (25 x 75 x 1 mm)	Fisher Scientific	12-550-413
Glass petri dish	Corning	3160-100BO
Glycerol	Sigma-Aldrich	G5516
Image Studio software version 5.2.5	LI-COR Biosciences		Image processing software for quantitation of Western blots.
Laemmli sample buffer	Bio-Rad	161-0737	2X concentrated protein sample buffer, supplement with beta mercaptoethanol as per manufacturer's instructions.
Lane marker reducing sample buffer	ThermoFisher Scientific	39000	5X concentrated protein sample buffer.
Microcentrigure tubes	Axygen	MCT-175-C
Microdissection scissors	Fine Science Tools	15000-03
Microwell trays (72 x 10 µL wells)	Nunc	438733
Mounting media			0.5% N-propylgallate and 80% glycerol in PBS
N-propylgallate	Sigma-Aldrich	P3130
Nuclease-free PBS (PBS in 0.1% DEPC, pH 7.4)			Add appropriate volume of DEPC to PBS, mix well and incubate overnight at room temperature with constant stirring. Autoclave for at least 20 minutes. Store at 4°C
PBS (phosphate buffered saline pH 7.4)	Sigma-Aldrich	P5368	Prepare according to manufacturer's instructions
PBS+pi (PBS plus protease and phoshatase inhibitors)			10mM NaF, 1mM beta-glycerol phosphate and 1mM Na3VO4 in PBS, pH 7.4.
PBT			0.15% TritonX and 0.5% bovine serum albumin in PBS, pH 7.4
Pin holder	Fine Science Tools	26016-12
Primary antibody: goat anti-GAPDH	Imgenex	IMG-3073	For Western blotting. Used at 1:3000
Primary antibody: rabbit anti-cleaved Dcp-1	Cell signaling	9578S	For immunofluorescence. Used at 1:100
Primary antibody: rat anti-DEcad	Developmental Studies Hybridoma Bank	DCAD2	For immunofluorescence. Used at 1:20
Primary antibody: rat anti-DEcad	DOI: 10.1006/dbio.1994.1287	DCAD1	Gift from Tadashi Uemura. Used at 1:100.
RNA extration kit: Relia Prep RNA tissue Miniprep kit	Promega	Z6110
Rnase decontamination reagent (RNase Away)	Molecular BioProducts	7002
Scalpel blades	Fine Science Tools	10050	Break off small piece of scapel blade and secure in blade holder.
Secondary antibody: 488-conjugated  donkey anti-rat IgG (H+L)	Jackson ImmunoResearch	712-545-153	For immunofluorescence. Used at 1:200
Secondary antibody: cy3-conjugated goat anti-rabbit IgG (H+L)	Jackson ImmunoResearch	111-165-144	For immunofluorescence. Used at 1:100
Secondary antibody: HRP-conjugated goat anti-rat IgG (H+L)	Cell Signaling Technology	7077	For Western blotting. Used at 1:3000
Secondary antibody: HRP-conjugated rabbit anti-goat IgG (H+L)	Jackson ImmunoResearch	305-035-003	For Western blotting. Used at 1:3000
Sodium Chloride	Sigma-Aldrich	S3014
Sodium Fluoride	Sigma-Aldrich	215309
Sodium vanadate	Sigma-Aldrich	50860
Spectrophotometer (NanoDrop)	ThermoFisher Scientific	2000c
Stereo dissecting microscope (M60 or M80)	Leica Microsystems		or similar microscope
Sylgard (black)	Dow Corning	SYLG170
Sylgard (transparent)	Dow Corning	SYLG184	Color black with finely ground charcol powder
Tissue: Kimwipes	KIMTECH	34120
TritonX	Sigma-Aldrich	T8787
Trizma hydrochloride pH7.5	Sigma-Aldrich	T5941
Tungsten needle, fine	Fine Science Tools	10130-10	Insert into pin holder
Tungsten needle, sturdy	Fine Science Tools	10130-20	Insert into pin holder
WTLB (western tissue lysis buffer)			150mM NaCl, 1.5% Triton X-100, 1mM EDTA, 20% glycerol, 10mM NaF, 1mM beta-glycerol phosphate and 1mM Na3VO4 in 50mM Tris-HCl (pH 7.5). Supplement with one cOmplete protease cocktail table per 10 mL solution.
Yeast paste	(local supermarket)		Approximately 2 tablespoons Fleischmann's ActiveDry Yeast (or similar) dissolved in ~20 mL distilled H2O