Sonication-facilitated Immunofluorescence Staining of Late-stage Embryonic and Larval Drosophila Tissues In Situ

Summary

Immunostaining is an effective technique for visualizing specific cell types and proteins within tissues. By utilizing sonication, the protocol described here alleviates the need to dissect Drosophila melanogaster tissues from late-stage embryos and larvae before immunostaining. We provide an efficient methodology for the immunostaining of formaldehyde-fixed whole mount larvae.

Abstract

Studies performed in Drosophila melanogaster embryos and larvae provide crucial insight into developmental processes such as cell fate specification and organogenesis. Immunostaining allows for the visualization of developing tissues and organs. However, a protective cuticle that forms at the end of embryogenesis prevents permeation of antibodies into late-stage embryos and larvae. While dissection prior to immunostaining is regularly used to analyze Drosophila larval tissues, it proves inefficient for some analyses because small tissues may be difficult to locate and isolate. Sonication provides an alternative to dissection in larval Drosophila immunostaining protocols. It allows for quick, simultaneous processing of large numbers of late-stage embryos and larvae and maintains in situ morphology. After fixation in formaldehyde, a sample is sonicated. Sample is then subjected to immunostaining with antigen-specific primary antibodies and fluorescently labeled secondary antibodies to visualize target cell types and specific proteins via fluorescence microscopy. During the process of sonication, proper placement of a sonicating probe above the sample, as well as the duration and intensity of sonication, is critical. Additonal minor modifications to standard immunostaining protocols may be required for high quality stains. For antibodies with low signal to noise ratio, longer incubation times are typically necessary. As a proof of concept for this sonication-facilitated protocol, we show immunostains of three tissue types (testes, ovaries, and neural tissues) at a range of developmental stages.

Introduction

Drosophila embryos and larvae provide an excellent model to study developmental processes in many organs and tissues. Imaging of individual cells is often necessary in these studies in order to ascertain the complex environments in which cells develop. Visualization of cells in tissues can be accomplished through immunostaining. Well-described immunostaining protocols exist for embryonic Drosophila tissues <17 hr after egg laying (AEL)^1-3. However, a protective cuticle forms toward the end of embryogenesis, preventing effective antibody permeation. Thus, these immunostaining protocols are inefficient in the analysis of tissues in late-stage embryos and in subsequent stages of larval development (1^st instar (L1), 2^nd instar (L2), and 3^rd instar (L3)). This inefficiency imposes a barrier to our understanding of dynamic processes that occur during this extended developmental period ⁴. Tissue dissection is a widely employed technique to circumvent this barrier ^5-7. However, dissection can prove inefficient. Extraction may be encumbered by difficulty in locating or isolating embryonic and larval tissues. Furthermore, the physical removal of target tissues may cause damage by rupturing them or by failing to extract them in their entirety.

Sonication is a method that employs sound waves to disturb intermolecular interactions. It has been used to disrupt the integrity of the Drosophila larval cuticle in order to immunostain developing neural cell types ⁶. This protocol has been adapted to immunostain late-stage embryonic and larval gonads, which can be as small as 50μm in diameter ^8-10. Through such studies, the process of male germline stem cell (GSC) niche formation has been characterized in late stage Drosophila embryos^8-10 and mechanisms regulating stem cell development and differentiation in late stage embryonic gonads and larvae have been elucidated ^9-12. Thus, sonication provides an efficient alternative to tissue dissection that may be difficult because of tissue size. Furthermore, it enables immunostaining of Drosophila tissues in situ, leaving cells within the context of the entire organism and maintaining in situ morphology. Here, we describe a step-by-step protocol for fluorescence immunostaining of late-stage embryonic through early/mid-L3 tissues in situ. Analysis of Drosophila gonadal and neural tissue is shown in the Representative Results to demonstrate the efficacy of this protocol. Furthermore, this immunostaining protocol may be adapted to analyze other Drosophila tissues as well as tissues in other organisms with an outer cuticle.

Protocol

1. Preparation of a Collection Cage Anesthetize young, fertile flies with CO2. Transfer anesthetized flies to a cage. To obtain optimal yield, use 100-120 adult flies ranging from 2-7 days of age at a 4:1 ratio of females to males. Allow flies an appropriate acclimation period, ~24 hr prior to obtaining sample for fixation. If the cage was set up with virgin females mated to males, a use a 36 – 48 hr acclimation period. On the open end of the cage, place a pre-prepared apple juice ag…

Representative Results

To demonstrate the efficacy of sonication-based immunostaining in analysis of late-stage embryonic and larval tissues in situ, wild-type embryos and larvae were processed for immunostaining of testes, ovaries, and neural tissue. Samples were imaged via confocal microscopy and representative results are shown (Figure 1 and Figure 2). Results reveal that the described protocol is effective for visualizing morphological features as well as individual cells in situ during l…

Discussion

This protocol provides a method to successfully immunostain target Drosophila embryonic and larval tissues in situ, thus eliminating the need for dissection. As per prior protocols for staining early embryos^1,2,3, the chorionic membrane is removed using 50% bleach (NaOCl). Samples are fixed in formaldehyde and methanol. Because the larval cuticle causes older sample to float, the entire sample is then passed through a cell-strainer to ensure larval retention. Sample is stored, if desired, in …

Materials

			Table 1: Reagents and Buffers
Phosphate Buffer Triton X-100 (PBTx)	For 5 L: 500 mL PBS 10X 4.45 L ddH2O 50 mL Triton 10%	Store at room temperature.
Phosphate Buffer Saline 10x (PBS 10X)	For 1 L in dH2O: 80 g NaCl 2 g KCl 14.4g Na2HPO4 2.4 g KH2PO4	Add components and fill to appropriate volume. Store at 4 degrees C.
Triton 10%	For 50 mL: 5 mL of Triton 5 mL of PBS 10X 45 mL of ddH2O	Rock to mix. Store at room temperature.
PEMS	0.1 M Pipes (pH 6.9) 2.0 mM MgSO4 1.0 mM EGTA	Store at room temperature.
Pipes	For a 400ml of a 0.25 M solution (pH 6.9): 30.24 g Pipes dH20 NaOH	Dissolve Pipes in 300 mL dH2Oand then adjust to pH 6.9 with NaOH. Bring the total volume to 400 mL with dH2O and autoclave. Store at room temperature.
Formaldehyde	37% formaldehyde by weight in methanol	Store at room temperature. Store formaldehyde, heptane, and methanol waste mixture in a tightly sealed container in fume hood before disposal as per institutional guidelines.
Heptane	n-Heptane	CAS 142-82-5. Store at room temperature. Store formaldehyde, heptane, and methanol waste mixture in a tightly sealed container in fume hood before disposal as per institutional guidelines.
Methanol	Methanol	CAS 67-56-1. Store at room temperature. Store formaldehyde, heptane, and methanol waste mixture in a tightly sealed container in fume hood before disposal as per institutional guidelines.
Phosphate Buffer Tween (PBTw)	To make 1 L: 100 mL PBS 10X 890 mL ddH2O 10 mL Tween 10%	Filter sterilize after adding all components. Store at 4 degrees C.
Tween 10%	For 50 mL: 5 mL Tween 5 mL of PBS 10X 40 mL of ddH2O	Rock to mix. Store at room temperature.
Bovine Serum Albumin/Phosphate Buffer Tween (BBTw)	To make 1 L: 100 mL PBS 10X 890 mL ddH2O 10 mL Tween 10% 1 g Bovine Serum Albumin (BSA)	Add BSA then sterilize using a 0.2 micrometer vacuum filter unit. Store at 4 degrees C.
Normal Goat Serum (NGS)	To make 10 mL: Normal Goat Serum (Jackson ImmunoResearch Laboratories Code: 005-000-121) 10 mL ddH2O	Add ddH2O to vial of NGS and sterilize using a 0.2 micrometer syrninge filter. Store aliquots at -20 degrees C.
1,4-diazabicyclo[2.2.2]octane (DABCO)	To make 100 mL: 25 mL ddH2O 1 mL Tris HCl (1M, pH 7.5) 2.5 g of DABCO solid (CAS: 281-57-9) 3.5 mL 6N HCl 250 uL 10N NaOH 70 mL glycerol	In 250 mL beaker with stir bar, add ddH2O, Tris HCl and DABCO. Stir and then add 6N HCl, 10 N NaOH, and glycerol. Then add 10NaOH dropwise until solution reaches pH 7.5. Aliquot. Store aliquots at -20 degrees C.
DABCO + p-phenylenediamine (PPD) Solution	1.765 mL NaHCO3 0.353 Na2CO3 0.02 g PPD (CAS: 106-50-3)	Dissolve PPD in NaHCO3 and NaCO3 solution. Add 60 uL of PPD solution to 500 uL of DABCO. Store aliquots at -20 degrees C.
Apple juice plates	To make ~200 plates: 45 g agar (CAS#9002-18-0) 45 g granulated sugar (store bought) 500 mL Apple juice (store bought) 15 mL Tegosept 10% 1.5 mL ddH2O	Add agar to ddH2O in 4L flask then autoclave for 30 minuntes. Mix apple juice and sugar on heated stir plate. Gradually add apple juice mixture to autoclaved agar. Mix on heated stir plate then aliquot 10mL volumes into 35 mm petri dishes and let stand at room temperature to solidfy. Store at 4 degrees C.
Tegosept 10%	To make 100mL: 10g Tegosept 100 mL Ethanol	Store aliquots at -20 degrees C
Yeast paste	~50 g Dry Active Yeast	Gradually add ddH2O to beaker containing yeast while stirring until paste-like consistency reached. Store at 4 degrees C.
			Table 2: Staining Materials
DAPI	1:1000	Invitrogen	D3571 //// Stock at 5mg/mL
rabbit anti-Vasa	1:250		A gift from Ruth Lehmann
mouse anti-Fasciclin III	1:10	Developmental Studies Hybridoma Bank (DSHB)	7G10
mouse anti-1B1	1:4	Developmental Studies Hybridoma Bank (DSHB)	1B1
guniea pig anti-Traffic Jam	1:2500		A gift from Dorthea Godt (Li et al, 2003)
mouse anti-Prospero	1:10	Developmental Studies Hybridoma Bank (DSHB)	Prospero MR1A
rat anti-Elav	1:30	Developmental Studies Hybridoma Bank (DSHB)	Rat-elav 7EBA10 anti-elav
mouse anti-Repo	1:10	Developmental Studies Hybridoma Bank (DSHB)	8D12 anti-Repo
goat anti-rabbit Alexa546	1:500	Invitrogen	A11010
goat anti-mouse Alexa488	1:500	Invitrogen	A11029
goat anti-guniea pig Alexa633	1:500	Invitrogen	A21105
goat anti-rat Alexa488	1:500	Invitrogen	A11006
			Table 3: Materials and Equipment
Fly Cages	Hand-made; Genesee Scientific Corporation	Not applicable; Bottles: 32-130; Pre-made cage: 59-101	Made by cutting clear cast acrylic tubing (1 3/4 inch in diameter) into 4 inch tall segments with a compound miter saw at 400 rpm. Ultrafine stainless steel screening (was attached to one end of the tub with acrylic compund glue. An alternate method using an empty fly food bottle can be found in Drosophila Protocols ISBN 0-87969-584-4. Cages may also be purchased from the Genesee Scientific Corporation.
Sonicator: Branson 250 Digital Sonifier	Branson	Model: Branson Digital Sonifier 250
Sonicator Probe	Branson	Model #: 102C (CE)	EDP: 101-135-066; S/N: OBU06064658
Syringe filter	Nalgene	190-25-20	0.2 micrometer cellulose, acetate membrane filter
Imaging system: Spinning disc confocal microscope with multichromatic light source, digital CCD camera, and imaging software	Microscope: Olympus Light source: Lumen Dynamics Camera: Q-Imaging Imaging Software: Intelligent Imaging Inc.	Microscope: BX51 equipped with DSU spinning disc Light source: X-Cite 120Q Camera: RETIGA-SRV Imaging Software: Slidebook 5.0
Vacuum filter unit	Nalgene	450-0020	0.2 micrometer cellulose nitrate membrane filter