Yuki X. Chen, Kelly Veerasammy, and Mayan Hein are supported by the Sloan Foundation CUNY Summer Research Program (CSURP). Jun Yin is supported by the intramural research program of the National Institutes of Health Project Number 1ZIANS003137. Support for this project was provided by a PSC-CUNY Award to Ye He and Rinat Abzalimov, jointly funded by The Professional Staff Congress and The City University of New York.

1. Fly head embedding
NOTE: The whole procedure takes ~45-60 min.
<ol>
	<li>Prepare the optimal cutting temperature compound (OCT compound) stage with a flat surface.
	<ol>
		<li>Add OCT into a plastic cryomold (15 mm x 15 mm x 5 mm) to half of the depth of the cryomold and avoid bubble formation. Leave the mold on a flat surface for several minutes, and then transfer it onto dry ice.</li>
		<li>Keep the cryomold flat on the dry ice and allow the OCT to form a flat and even surface. Wait until the OCT is fully solidified in the mold. Use the frozen OCT stages immediately or store them at &#8722;80 &#176;C.</li>
	</ol>
	</li>
	<li>Anesthetize adult flies using CO2 (i.e., a CO2 pad).
	<ol>
		<li>Prepare a Petri dish containing a piece of laboratory wipe. Use water to moisten part of the wipe to reduce the static electricity. Keep the wipe half wet and half dry.</li>
		<li>Under dissecting scope 1, use forceps to cut the fly head. Collect 4-5 heads each time and put them onto the dry area of the laboratory wipe. 
		NOTE: The collection of 4-5 heads takes ~ 2 mins.</li>
	</ol>
	</li>
	<li>Transfer the OCT stage from dry ice to the dissection scope.
	<ol>
		<li>Take the OCT stage from dry ice to microscope 2, immediately transfer the heads to the OCT stage, and arrange them quickly, which takes ~30 s to avoid OCT melting. Leave ~1 mm of blank space around each fly brain to ensure adequate support from the OCT and 4-5 mm of blank space from the edge of the block to provide adequate room to handle the section. If the proboscis is too long, remove the tip; if it is not too long, keep it intact. Put the OCT stage back onto the dry ice and let it stay on for ~3 min to make sure the OCT stage remains frozen and solid.</li>
		<li>When the OCT stage is back on the dry ice and waiting for solidification, collect another round of heads. Repeat steps 1.2 and 1.3 to transfer additional samples onto the remaining space on the stage. 
		NOTE: Eight heads are usually prepared for each genotype, and four genotypes are used in one OCT stage in this laboratory.</li>
		<li>Use two dissection microscopes, side by side, to avoid changing focus and increasing the time taken to transfer and arrange the heads on the OCT stage and to lower the risk of the OCT stage melting.</li>
	</ol>
	</li>
	<li>After all the fly heads are aligned, let the OCT stage sit on the dry ice for another 5-10 min.</li>
	<li>Take the OCT stage away from the dry ice, put it on a flat surface (bench), and then, quickly, add a large amount of OCT compound to cover all the samples and fill the whole cryomold, which takes ~3 s.</li>
	<li>Immediately transfer the cryomold back to dry ice and freeze the whole OCT block containing the embedded tissues. Let the OCT stage sit on the dry ice for another 5-10 min. Label the samples on the margin of the cryomold.</li>
	<li>Store the frozen samples at &#8722;80 &#176;C until ready for sectioning.</li>
</ol>
2. Cryosectioning the tissue
NOTE: When handling the indium tin oxide (ITO) slides, wear gloves at all times to avoid tissue contamination. Wearing a mask is also recommended to avoid breathing directly onto the slide.
<ol>
	<li>Confirm the ITO-coated side by testing the conductivity of the ITO slides using a voltmeter set to resistance. Mark the side with a resistance measurement as the side to adhere the tissue to. Label it and always set a laboratory wipe on the bottom of the slide to avoid slide contamination.</li>
	<li>Allow the tissues to equilibrate in the cryostat chamber for 30-45 min.
	<ol>
		<li>To avoid the melting of the OCT, place all the necessary tools such as forceps and a thin-tipped artist brush in the cryostat chamber ahead of time to precool them.</li>
	</ol>
	</li>
	<li>Clean the cryostat, preferably with 70% ethanol. Wipe the roll plate and stage and remove used blades. Use additional clean wipes to ensure that the ethanol has evaporated and that all the surfaces are dry before sectioning begins.</li>
	<li>Adjust the temperature of the cryostat chamber and specimen head according to the type of the tissue (e.g., &#8722;14 &#176;C for liver, &#8722;20 &#176;C for muscle, and &#8722;25 &#176;C for skin10; &#8722;18 &#176;C for fly heads in this protocol).</li>
	<li>Mount the tissue onto the specimen holder using OCT. Be careful to use enough OCT to cover the base of the OCT block and mount the block as flat as possible.</li>
	<li>Place a clean blade in the stage and lock it. Position the head of the specimen toward the stage as needed to achieve the desired cutting angle.
	<ol>
		<li>Place the specimen block in an orientation where all genotypes/treatment groups are positioned vertically to the blade. 
		NOTE: This ensures a consistent cutting plane and avoids cross-contamination from different groups. If cutting a different block of tissue, switch to a new blade between samples to prevent cross-contamination.</li>
	</ol>
	</li>
	<li>Begin cutting in thick sections (50-100 &#181;m) until the region of interest (e.g., the desired region of the brain) is found.
	<ol>
		<li>Constantly brush off extra pieces with a precooled artist brush to keep the stage clean.</li>
	</ol>
	</li>
	<li>Change the thickness of the sections to 10-12 &#181;m once the desired region is reached.
	<ol>
		<li>Adjust the chamber temperature slightly as necessary. For example, set a higher temperature if the section tends to flake or fall apart easily. 
		NOTE: The recommended temperature for OCT blocks is &#8722;18 &#176;C.</li>
	</ol>
	</li>
	<li>Carefully collect the desired section and adhere it to the ITO slide. Perform this operation in the cryostat chamber.
	<ol>
		<li>Take a room-temperature ITO slide and position it over the section, approach&#160;the section gently and quickly for the section to adhere to the slide without leaving traces on the cryostat stage. 
		NOTE: The OCT will melt and cling to the slide.</li>
	</ol>
	</li>
	<li>Place the slide aside in a rack or laboratory wipe outside the cryostat between the collections of multiple sections.</li>
	<li>If comparison across different samples of the same cohort is desired, place the sections from multiple samples onto a single slide for simultaneous analysis and minimal&#160;variation. If necessary, separate to two slides, as the MALDI target holder can accommodate two slides in a single run.</li>
	<li>Transport the slides in a vacuum box to a desiccator with desiccant as the bottom layer. Dry the slides under a vacuum for 30-60 min. 
	NOTE: Alternatively, if the lab is not equipped with a vacuum desiccator, the slides should be kept at &#8722;20 &#176;C throughout the process until storage at &#8722;80 &#176;C within 24 h or shipping on dry ice to avoid the deterioration of lipids or metabolites.</li>
	<li>Proceed to matrix deposition. Use 2,5-dihydroxybenzoic acid (DHB) in methanol/water (70/30, v/v) as the matrix.</li>
	<li>If slides are not run immediately, either store the slides at &#8722;80 &#176;C (up to 1 month for fly brain sections and 6 months for rodent brain sections), or immediately ship the sample to MALDI core facilities with adequate dry ice. For optimal storage and shipping, place the slides into a slide transporter and securely seal the opening with wax film. Seal with a zip bag, place it into another zip bag containing desiccant, and label the outside. 
	NOTE: Refer to previous work for the followed steps of slide scanning, matrix deposition, and MALDI imaging procedures11.</li>
	<li>Perform matrix deposition using the automatic HTX M5 matrix sprayer and a 40 mg/mL solution of DHB in methanol/water (70/30, v/v). Spray the matrix at a customized flow rate of 0.12 mL/min and a nozzle temperature of 85 &#176;C for 10 passes. Use an N2 gas pressure of 10 psi. 
	NOTE: If the N2 gas pressure in the sprayer is lowered below 5 psi, a safety mechanism in the sprayer will shut the heater off at once to avoid any damage to the sprayer with a 1,300 mm/min spray velocity, 2 mm track spacing, 3 L/min flow rate, and 40 mm nozzle height.</li>
	<li>Use a MALDI time of flight (TOF) MS instrument (see the Table of Materials) in positive ion mode to acquire a mass spectrum within the mass ranges from m/z 50-1,000.
	<ol>
		<li>To calibrate the instrument, spot 0.5 &#181;L of red phosphorus emulsion in acetonitrile onto the ITO slides and use its spectra to calibrate the instrument in the 50-1,000 m/z mass range by applying a quadratic calibration curve2.</li>
		<li>Set the laser spot diameters to Medium, as it is the modulated beam profile for 40 &#181;m raster width, and gather imaging data by summing 500 shots at a laser repetition rate of 1,000 Hz per array position.</li>
		<li>Use software (see the Table of Materials) to record and process the spectral data. Perform imaging data analysis using root mean square (RMS) normalization to generate ion images at a bin width of &#177;0.10 Da. Align the spectra of both the OCT and brain tissue from the same experiment using the software to evaluate the overlapping peaks and the ion suppression interference of the OCT (Supplemental Figure S1). After the experiment, process the MALDI slides containing the tissue samples by hematoxylin and eosin (H&#38;E) staining, as previously described11.</li>
	</ol>
	</li>
</ol>

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The authors have no conflicts of interest to disclose.

As demonstrated in the study on the variations in lipid composition in mutant and wild-type Drosophila brains, MALDI MSI can be a valuable label-free imaging technique for in situ analysis of molecular distribution patterns within organs of small insects. Indeed, because lipids are distributed in both the brain tissue and fat bodies of Drosophila heads, conventional lipidomics approaches based on liquid-chromatography and mass-spectrometry (LC-MS) can detect only combined signals from both regi...

Lipids are involved in a wide range of biological processes and can be broadly classified into five categories based on their structural diversity: fatty acids, triacylglycerols (TAGs), phospholipids, sterol lipids, and sphingolipids1. The fundamental functions of lipids are to provide energy sources for biological processes (i.e., TAGs) and form cellular membranes (i.e., phospholipids and cholesterol).&#160;However, additional roles of lipids have been noted in development and diseases, and have been extensively studied in the biomedical field. For instance, reports have shown that fatty acids of different lengths may have unique therapeutic roles. Short fatty acid chains can be involved in defense mechanisms against autoimmune diseases, medium-length fatty acid chains produce metabolites that can mitigate seizures, and long fatty acid chains generate metabolites that can be used to treat metabolic disorders2. In the nervous system, glia-derived cholesterol and phospholipids have been shown to be vital for synaptogenesis3,4. Other types of lipids have shown promise in medical applications, including&#160;sphingolipids utilized in drug delivery systems and saccharolipids&#160;used to support the immune system5,6. The numerous roles and potential therapeutic applications of lipids in the biomedical field have made lipidomics&#8212;the study of the pathways and interactions of cellular lipids&#8212;a critical and increasingly important field.
Lipidomics makes use of analytical chemistry to study the lipidome on a large scale. The main experimental methods utilized in lipidomics are based on mass spectrometry (MS) coupled with various chromatography and ion-mobility techniques7,8. The use of MS in the area is advantageous due to its high specificity and sensitivity, speed of acquisition, and unique capabilities to (1) detect lipids and lipid metabolites occurring even at low and transient levels, (2) detect hundreds of different lipid compounds in a single experiment, (3) identify previously unknown lipids, and (4) distinguish between lipid isomers. Among the developments in MS, including desorption electrospray ionization (DESI), MALDI, and secondary ion mass spectrometry (SIMS), MALDI MSI has emerged as a powerful imaging technique that complements conventional MS-based approaches by providing unique information on the spatial distribution of lipids within tissue compartments9,10.
The typical workflow of lipidomics consists of sample preparation, data acquisition using mass-spectrometry technology, and data analysis11. The study of lipids and metabolites in samples has led to the emergence of techniques to understand the physiological and pathological conditions of metabolic processes in organisms. While understanding biological interactions is important, the sensitivity of lipids and metabolites makes them difficult to image and identify without dyes or other modification. Changes in metabolite levels or distribution may lead to phenotypic changes. One tool used for metabolomic profiling is MALDI MSI, a label-free, in situ imaging technique capable of detecting hundreds of molecules simultaneously. MALDI imaging allows for the visualization of metabolites and lipids in samples while preserving their integrity and spatial distribution. Previous technology for lipid profiling involved the use of radioactive chemicals to individually map lipids, while MALDI imaging forgoes this and allows for the detection of a range of lipids simultaneously.
Lipid metabolism and homeostasis play important functions in cell physiology, such as the maintenance and development of the nervous system. One essential aspect of nervous system lipid metabolism is the lipid shuttling between neurons and glial cells, which is mediated by molecular carrier lipoproteins, including very-low-density lipoprotein (VLDL), low-density lipoproteins (LDL), and high-density lipoproteins (HDL)12. Lipoproteins contain apolipoproteins (Apo), such as ApoB and ApoD, which function as structural blocks of lipid cargo and as ligands for lipoprotein receptors. The neuron-glia crosstalk of lipids involves multiple players such as glia-derived ApoD, ApoE, and ApoJ, and their neuronal LDL receptors (LDLRs)13,14. In Drosophila, apolipophorin, a member of the ApoB family, is a major hemolymph lipid carrier15. Apolipophorin has two closely related lipophorin receptors (LpRs), LpR1 and LpR2, which are homologs of mammalian LDLR15,16. In previous studies, the astrocyte-secreted lipocalin Glial Lazarillo (GLaz), a Drosophila homolog of human ApoD, and its neuronal receptor LpR1 were discovered to cooperatively mediate neuron-glia lipid shuttling, thus regulating dendrite morphogenesis17. Therefore, it was speculated that the loss of LpR1 would cause a decrease in overall lipid content in the Drosophila brain. MALDI MSI would be a suitable tool for profiling the lipid contents in small tissues of LpR1&#8722;/&#8722; mutant and wild-type Drosophila brains, as demonstrated in this study.
Despite the growing popularity of MALDI MSI, the instrument&#39;s high cost and experimental complexity often impede its implementation in individual laboratories. Thus, most MALDI MSI studies are conducted using shared core facilities. As with other applications of MALDI MSI, a careful sample preparation process for lipidomics is critical to achieve reliable results. However, because sample slide preparation is typically performed in individual research laboratories, there is a possibility of variation in MALDI MSI acquisition. To combat this, this paper aims to provide a detailed protocol for the sample preparation of small biological samples prior to MALDI MSI measurement using lipid analysis of a large group of adult Drosophila brains in positive ion mode as an example11,17. However, some phospholipid classes and the majority of small metabolites are favorably detected by MALDI imaging in negative ion mode, which was described previously11. Therefore, with these two example studies, we hope to provide detailed sample preparation protocols of various combinations: free-standing large tissue versus embedded small tissue, thaw-mounting versus warm-slide mounting, and positive ion mode versus negative ion mode.

<table><tbody><tr><td>2,5-Dihydroxybenzoic acid (DHB)</td><td>Millipore Sigma Aldrich</td><td>85707-1G-F</td><td></td></tr><tr><td>Andwin Scientific&amp;nbsp;CRYOMOLD 15X15X5</td><td>Fisher Scientific</td><td>NC9464347</td><td></td></tr><tr><td>Andwin Scientific&amp;nbsp;Tissue-Tek CRYO-OCT Compound</td><td>Fisher Scientific</td><td>14-373-65</td><td></td></tr><tr><td>Artist brush MSC #5 1/8 X 9/16 TRIM RED SABLE</td><td>Fisher Scientific</td><td>50-111-2302</td><td></td></tr><tr><td>autoflex speed MALDI-TOF MS system</td><td>Bruker Daltonics Inc</td><td></td><td>MALDI-TOF MS instrument</td></tr><tr><td>BD&amp;nbsp;Syringe with Luer-Lok Tips</td><td>Fisher Scientific</td><td>14-823-16E</td><td></td></tr><tr><td>BD&amp;nbsp;Vacutainer General Use Syringe Needles</td><td>Fisher Scientific</td><td>23-021-020</td><td></td></tr><tr><td>Bruker Daltonics&amp;nbsp;GLASS SLIDES MALDI IMAGNG</td><td>Fisher Scientific</td><td>NC0380464</td><td></td></tr><tr><td>Drierite, with indicator, 8 mesh, ACROS Organics</td><td></td><td>AC219095000</td><td></td></tr><tr><td>Epson Perfection V600 Photo Scanner</td><td>Amazon</td><td>Perfection V600</td><td></td></tr><tr><td>Fisherbrand&amp;nbsp;5-Place Slide Mailer</td><td>Fisher Scientific</td><td>HS15986</td><td></td></tr><tr><td>Fisherbrand&amp;nbsp;Digital Auto-Range Multimeter</td><td>Fisher Scientific</td><td>01-241-1</td><td></td></tr><tr><td>FlexImaging v3.0</td><td>Bruker Daltonics Inc</td><td></td><td>Bruker MS imaging analysis software</td></tr><tr><td>HPLC Grade Methanol</td><td>Fisher Scientific</td><td>MMX04751</td><td></td></tr><tr><td>HPLC Grade Water</td><td>Fisher Scientific</td><td>W5-1</td><td></td></tr><tr><td>HTX M5 Sprayer</td><td>HTX Technologies, LLC</td><td></td><td>Automatic heated matrix sprayer</td></tr><tr><td>Kimberly-Clark Professional&amp;nbsp;Kimtech Science Kimwipes Delicate Task Wipers</td><td>Fisher Scientific</td><td>06-666A</td><td></td></tr><tr><td>MSC&amp;nbsp;Ziploc Freezer Bag</td><td>Fisher Scientific</td><td>50-111-3769</td><td></td></tr><tr><td>SCiLS Lab (2015b)</td><td>SCiLS Lab</td><td></td><td>Advanced MALDI MSI data analysis software</td></tr><tr><td>Thermo Scientific&amp;nbsp;CryoStar NX50 Cryostat</td><td>Fisher Thermo Scientific</td><td>95-713-0</td><td></td></tr><tr><td>Thermo Scientific&amp;nbsp;Nalgene Transparent Polycarbonate Classic Design Desiccator</td><td>Fisher Scientific</td><td>08-642-7</td><td></td></tr></tbody></table>

sample preparation for rapid lipid analysis in drosophila brain using matrix-assisted laser desorption/ionization mass spectrometry imaging

Lipid profiling, or lipidomics, is a well-established technique used to study the entire lipid content of a cell or tissue. Information acquired from&#160;lipidomics is valuable in studying the pathways involved in development, disease, and cellular metabolism. Many tools and instrumentations have aided lipidomics projects, most notably various combinations of mass spectrometry and liquid chromatography techniques. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) has recently emerged as a powerful imaging technique that complements conventional approaches. This novel technique provides unique information on the spatial distribution of lipids within tissue compartments, which was previously unattainable without the use of excessive modifications. The sample preparation of the MALDI MSI approach is critical and, therefore, is the focus of this paper. This paper presents a rapid lipid analysis of a large number of Drosophila brains embedded in optimal cutting temperature compound (OCT) to provide a detailed protocol for the preparation of small tissues for lipid analysis or metabolite and small molecule analysis through MALDI MSI.

The loss of the neuronal receptor LpR1 of the astrocyte-secreted lipocalin Glial Lazarillo (GLaz), a Drosophila homolog of human ApoD, was hypothesized to be able to cause a decrease in overall lipid content in the Drosophila brain. To test this, MALDI MSI was used to profile the lipids in LpR1&#8722;/&#8722; mutant and wild-type Drosophila brains, which is elaborated on below.
The experiment was performed according to the workflow shown in <stron...

Watch this Scientific Journal Video about Sample Preparation for Rapid Lipid Analysis in Drosophila Brain Using Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging at JoVE.com

Sample Preparation for Rapid Lipid Analysis in Drosophila Brain Using Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging

The aim of this protocol is to provide detailed guidance on the proper sample preparation for lipid and metabolite analysis in small tissues, such as the Drosophila brain, using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging.

Sample Preparation for Rapid Lipid Analysis in Drosophila Brain Using Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging

Lipid profiling, or lipidomics, is a well-established technique used to study the entire lipid content of a cell or tissue. Information acquired ...

sample-preparation-for-rapid-lipid-analysis-drosophila-brain-using

Research

JoVE Journal

Biology

3.1K Views.  the City University of New York, Graduate Center New York. Maldi MS-spectrometrie beeldvorming is een gevestigd en nuttig hulpmiddel om lipidenrijkdom en -distributie te identificeren en te visualiseren, zonder overmatige monstermodificatie. Het belangrijkste voordeel van Maldi-beeldvorming is het vermogen om de lipiden in situ te detecteren zonder etikettering en tegelijkertijd grote populatie monsters te analyseren. Oefening baart kunst.Zorg ervoor dat je goed oefent voordat je de echte experimentele monsters verwerkt. Minimaliseer de voorbe...

Video: Sample Preparation for Rapid Lipid Analysis in Drosophila Brain Using Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging

Laser Capture Microdissection of Drosophila Peripheral Neurons

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the molecular mechanisms underlying class-specific dendrite morphogenesis1,2. To facilitate molecular analyses of class-specific da neuron development, it is vital to obtain these cells in a pure population. Although a range of different cell, and tissue-specific RNA isolation techniques exist for Drosophila cells, including magnetic bead based cell purification3,4, Fluorescent Activated Cell Sorting (FACS)5-8, and RNA binding protein based strategies9, none of these methods can be readily utilized for isolating single or multiple class-specific Drosophila da neurons with a high degree of spatial precision. Laser Capture Microdissection (LCM) has emerged as an extremely powerful tool that can be used to isolate specific cell types from tissue sections with a high degree of spatial resolution and accuracy. RNA obtained from isolated cells can then be used for analyses including qRT-PCR and microarray expression profiling within a given cell type10-16. To date, LCM has not been widely applied in the analysis of Drosophila tissues and cells17,18, including da neurons at the third instar larval stage of development. 
Here we present our optimized protocol for isolation of Drosophila da neurons using the infrared (IR) class of LCM. This method allows for the capture of single, class-specific or multiple da neurons with high specificity and spatial resolution. Age-matched third instar larvae expressing a UAS-mCD8::GFP19 transgene under the control of either the class IV da neuron specific ppk-GAL420 driver or the pan-da neuron specific 21-7-GAL421 driver were used for these experiments. RNA obtained from the isolated da neurons is of very high quality and can be directly used for downstream applications, including qRT-PCR or microarray analyses. Furthermore, this LCM protocol can be readily adapted to capture other Drosophila cell types a various stages of development dependent upon the cell type specific, GAL4-driven expression pattern of GFP.

Laser Capture Microdissection of Drosophila Peripheral Neurons

In this video-article we present a method for isolating single or multiple Drosophila da neurons from third instar larvae using the infrared capture (IR) class of Laser Capture Microdissection (LCM). RNA obtained from the isolated neurons can be readily used for downstream applications including qRT-PCR or microarray analyses.

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the ...

Neuroscience

Lipid Droplet Isolation for Quantitative Mass Spectrometry Analysis

Lipid droplets are vital to the replication of a variety of different pathogens, most prominently the Hepatitis C Virus (HCV), as the putative site of virion morphogenesis. Quantitative lipid droplet proteome analysis can be used to identify proteins that localize to or are displaced from lipid droplets under conditions such as virus infections. Here, we describe a protocol that has been successfully used to characterize the changes in the lipid droplet proteome following infection with HCV. We use Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) and thus label the complete proteome of one population of cells with &#34;heavy&#34; amino acids to quantitate the proteins by mass spectrometry. For lipid droplet isolation, the two cell populations (i.e.&#160;HCV-infected/&#34;light&#34; amino acids and uninfected control/&#34;heavy&#34; amino acids) are mixed 1:1 and lysed mechanically in hypotonic buffer. After removing the nuclei and cell debris by low speed centrifugation, lipid droplet-associated proteins are enriched by two subsequent ultracentrifugation steps followed by three washing steps in isotonic buffer. The purity of the lipid droplet fractions is analyzed by western blotting with antibodies recognizing different subcellular compartments. Lipid droplet-associated proteins are then separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Coomassie staining. After tryptic digest, the peptides are quantified by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). Using this method, we identified proteins recruited to lipid droplets upon HCV infection that might represent pro- or antiviral host factors. Our method can be applied to a variety of different cells and culture conditions, such as infection with pathogens, environmental stress, or drug treatment.

Lipid droplets are important organelles for the replication of several pathogens, including the Hepatitis C Virus (HCV). We describe a method to isolate lipid droplets for quantitative mass spectrometry of associated proteins; it can be used under a variety of conditions, such as virus infection, environmental stress, or drug treatment.

Lipid droplets are vital to the replication of a variety of different pathogens, most prominently the Hepatitis C Virus (HCV), as the putative site of ...

Biochemistry

Single Drosophila Ommatidium Dissection and Imaging

The fruit fly Drosophila melanogaster has made invaluable contributions to neuroscience research and has been used widely as a model for neurodegenerative diseases because of its powerful genetics1. The fly eye in particular has been the organ of choice for neurodegeneration research, being the most accessible and life-dispensable part of the Drosophila nervous system. However the major caveat of intact eyes is the difficulty, because of the intense autofluorescence of the pigment, in imaging intracellular events, such as autophagy dynamics2, which are paramount to understanding of neurodegeneration.
We have recently used the dissection and culture of single ommatidia3 that has been essential for our understanding of autophagic dysfunctions in a fly model of Dentatorubro-Pallidoluysian Atrophy (DRPLA)3, 4.
We now report a comprehensive description of this technique (Fig. 1), adapted from electrophysiological studies5, which is likely to expand dramatically the possibility of fly models for neurodegeneration. This method can be adapted to image live subcellular events and to monitor effective drug administration onto photoreceptor cells (Fig. 2). If used in combination with mosaic techniques6-8, the responses of genetically different cells can be assayed in parallel (Fig. 2).

Single Drosophila Ommatidium Dissection and Imaging

The limiting factor in the use of the adult Drosophila eye to study neurodegeneration and cell biology is the difficult imaging of intracellular processes. We describe the dissection of single ommatidia to generate a bona-fide primary neuronal cell culture, which can be subject to drug treatment and advanced imaging.

The fruit fly Drosophila melanogaster has made invaluable contributions to neuroscience research and has been used widely as a model for neurodegenerative ...

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

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.

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

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.&#160;

The Drosophila pupal retina provides an excellent model system for the study of morphogenetic processes during development. In this paper, we present a ...

Developmental Biology

Assay for Blood-brain Barrier Integrity in Drosophila melanogaster

Proper nervous system development includes the formation of the blood-brain barrier, the diffusion barrier that tightly regulates access to the nervous system and protects neural tissue from toxins and pathogens. Defects in the formation of this barrier have been correlated with neuropathies, and the breakdown of this barrier has been observed in many neurodegenerative diseases. Therefore, it is critical to identify the genes that regulate the formation and maintenance of the blood-brain barrier to identify potential therapeutic targets. In order to understand the exact roles these genes play in neural development, it is necessary to assay the effects of altered gene expression on the integrity of the blood-brain barrier. Many of the molecules that function in the establishment of the blood-brain barrier have been found to be conserved across eukaryotic species, including the fruit fly, Drosophila melanogaster. Fruit flies have proven to be an excellent model system for examining the molecular mechanisms regulating nervous system development and function. This protocol describes a step-by-step procedure to assay for blood-brain barrier integrity during the embryonic and larval stages of D. melanogaster development.

Assay for Blood-brain Barrier Integrity in Drosophila melanogaster

Blood-brain barrier integrity is critical for nervous system function. In Drosophila melanogaster, the blood-brain barrier is formed by glial cells during late embryogenesis. This protocol describes methods to assay for blood-brain barrier formation and maintenance in D. melanogaster embryos and third instar larvae.

Proper nervous system development includes the formation of the blood-brain barrier, the diffusion barrier that tightly regulates access to the nervous ...

Analysis of Lipid Signaling in Drosophila Photoreceptors using Mass Spectrometry

The activation of phospholipase C&#946; (PLC&#946;) is an essential step during sensory transduction in Drosophila photoreceptors. PLC&#946; activity results in the hydrolysis of the membrane lipid phosphatidylinositol 4,5 bisphosphate [PI(4,5)P2] leading ultimately to the activation of transient receptor potential (TRP) and TRP like (TRPL) channels. The activity of PLC&#946; also leads subsequently to the generation of many lipid species several of which have been proposed to play a role in TRP and TRPL activation. In addition, several classes of lipids have been proposed to play key roles in organizing the cell biology of photoreceptors to optimize signaling reactions for optimal sensory transduction. Historically, these discoveries have been driven by the ability to isolate Drosophila mutants for enzymes that control the levels of specific lipids and perform analysis of photoreceptor physiology in these mutants. More recently, powerful mass spectrometry methods for isolation and quantitative analysis of lipids with high sensitivity and specificity have been developed. These are particularly suited for use in Drosophila where lipid analysis is now possible from photoreceptors without the need for radionuclide labeling. In this article, the conceptual and practical considerations in the use of lipid mass spectrometry for the robust, sensitive, and accurate quantitative assessment of various signaling lipids in Drosophila photoreceptors are covered. Along with existing methods in molecular genetics and physiological analysis such lipid is likely to enhance the power of photoreceptors as a model system for discoveries in biology.

Analysis of Lipid Signaling in Drosophila Photoreceptors using Mass Spectrometry

The manuscript presents versatile, robust, and sensitive mass spectrometry protocols to identify and quantify several classes of lipids from Drosophila photoreceptors.

The activation of phospholipase C&#946; (PLC&#946;) is an essential step during sensory transduction in Drosophila photoreceptors. PLC&#946; activity ...

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

This protocol describes a method for measuring the metabolism in Drosophila melanogaster larval and adult brains. Quantifying metabolism in whole organs provides a tissue-level understanding of energy utilization that cannot be captured when analyzing primary cells and cell lines. While this analysis is ex vivo, it allows for the measurement from a number of specialized cells working together to perform a function in one tissue and more closely models the in vivo organ. Metabolic reprogramming has been observed in many neurological diseases, including neoplasia, and neurodegenerative diseases. This protocol was developed to assist the D. melanogaster community&#39;s investigation of metabolism in neurological disease models using a commercially available metabolic analyzer. Measuring metabolism of whole brains in the metabolic analyzer is challenging due to the geometry of the brain. This analyzer requires samples to remain at the bottom of a 96-well plate. Cell samples and tissue punches can adhere to the surface of the cell plate or utilize spheroid plates, respectively. However, the spherical, three-dimensional shape of D. melanogaster brains prevents the tissue from adhering to the plate. This protocol requires a specially designed and manufactured micro-tissue restraint that circumvents this problem by preventing any movement of the brain while still allowing metabolic measurements from the analyzer&#39;s two solid-state sensor probes. Oxygen consumption and extracellular acidification rates are reproducible and sensitive to a treatment with metabolic inhibitors. With a minor optimization, this protocol can be adapted for use with any whole tissue and/or model system, provided that the sample size does not exceed the chamber generated by the restraint. While basal metabolic measurements and an analysis after a treatment with mitochondrial inhibitors are described within this protocol, countless experimental conditions, such as energy source preference and rearing environment, could be interrogated.

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

We present a protocol for measuring oxygen consumption and extracellular acidification in Drosophila melanogaster larval and adult brains. A metabolic analyzer is utilized with an adapted and optimized protocol. Micro-tissue restraints are a critical component of this protocol and were designed and created specifically for their use in this analysis.

This protocol describes a method for measuring the metabolism in Drosophila melanogaster larval and adult brains. Quantifying metabolism in whole organs ...

Lipidomics and Transcriptomics in Neurological Diseases

Lipids serve as the primary interface to brain insults or stimuli conducive to neurological diseases and are a reservoir for the synthesis of lipids with various signaling or ligand function that can underscore the onset and progression of diseases. Often changing at the presymptomatic level, lipids are an emerging source of drug targets and biomarkers. Many neurological diseases exhibit neuroinflammation, neurodegeneration, and neuronal excitability as common hallmarks, partly modulated by specific lipid signaling systems. The interdependence and interrelation of synthesis of various lipids prompts a multilipid, multienzyme, and multireceptor analysis in order to derive the commonalities and specificities of neurological contexts and to expedite the unravelling of mechanistic aspects of disease onset and progression. Ascribing lipid roles to distinct brain regions advances the determination of lipid molecular phenotype and morphology associated with a neurological disease.
Presented here is a modular protocol suitable for the analysis of membrane lipids and downstream lipid signals along with mRNA of enzymes and mediators underlying their functionality, extracted from discrete brain regions that are relevant for a particular neurological disease and/or condition. To ensure accurate comparative lipidomic profiling, the workflows and operating criteria were optimized and standardized for: i) brain sampling and dissection of regions of interest, ii) co-extraction of multiple lipid signals and membrane lipids, iii) dual lipid/mRNA extraction, iv) quantification by liquid chromatography multiple reaction monitoring (LC/MRM), and v) standard mRNA profiling. This workflow is amenable for the low tissue amounts obtained by sampling of the functionally discrete brain subregions (i.e. by brain punching), thus preventing bias in multimolecular analysis due to tissue heterogeneity and/or animal variability. To reveal peripheral consequences of neurological diseases and establish translational molecular readouts of neurological disease states, peripheral organ sampling, processing, and their subsequent lipidomic analysis, as well as plasma lipidomics, are also pursued and described. The protocol is demonstrated on an acute epilepsy mouse model.

This article presents a modular protocol for tissue lipidomics and transcriptomics, and plasma lipidomics in neurological disease mouse models targeting lipids underlying inflammation and neuronal activity, membrane lipids, downstream messengers, and mRNA-encoding enzymes/receptors underlying lipid function. Sampling, sample processing, extraction, and quantification procedures are outlined.

Lipids serve as the primary interface to brain insults or stimuli conducive to neurological diseases and are a reservoir for the synthesis of lipids with ...

Dithranol as a Matrix for Matrix Assisted Laser Desorption/Ionization Imaging on a Fourier Transform Ion Cyclotron Resonance Mass Spectrometer

Mass spectrometry imaging (MSI) determines the spatial localization and distribution patterns of compounds on the surface of a tissue section, mainly using MALDI (matrix&#160;assisted laser desorption/ionization)-based analytical techniques. New matrices for small-molecule MSI, which can improve the analysis of low-molecular weight (MW) compounds, are needed. These matrices should provide increased analyte signals while decreasing MALDI background signals. In addition, the use of ultrahigh-resolution instruments, such as Fourier transform ion cyclotron resonance (FTICR) mass spectrometers, has&#160;the ability to resolve analyte signals from matrix signals, and this can partially overcome many problems associated with the background originating from the MALDI matrix. The reduction in the intensities of the metastable matrix clusters by FTICR MS can also help to overcome some of the interferences associated with matrix peaks on other instruments. High-resolution instruments such as the FTICR mass spectrometers are advantageous as they can produce distribution patterns of many compounds simultaneously while still providing confidence in chemical identifications. Dithranol (DT; 1,8-dihydroxy-9,10-dihydroanthracen-9-one) has previously been reported as a MALDI matrix for tissue imaging. In this work, a protocol for the use of DT for MALDI imaging of endogenous lipids from the surfaces of mammalian tissue sections, by positive-ion MALDI-MS, on an ultrahigh-resolution hybrid quadrupole FTICR instrument has been provided.

Dithranol (DT; 1,8-dihydroxy-9,10-dihydroanthracen-9-one) has previously been reported as a MALDI matrix for tissue imaging of small molecules; protocols for the use of DT for the MALDI imaging of endogenous lipids on the surface of tissue sections&#160;by positive-ion MALDI-MS on an ultrahigh-resolution quadrupole-FTICR instrument are provided here.

Mass spectrometry imaging (MSI) determines the spatial localization and distribution patterns of compounds on the surface of a tissue section, mainly ...

Fluorescence-Guided Matrix-assisted Laser Desorption/Ionization with Laser-Induced Postionization Mass Spectrometry of Individual Rat Neural Cells

Single-cell measurements are critical to understanding the rich spatiochemical heterogeneity of the brain. Matrix-assisted laser/desorption ionization (MALDI) mass spectrometry (MS) is capable of label-free, high-throughput characterization of endogenous molecules in individual cells. The recent advances in the development of MALDI mass spectrometers with laser-induced post-ionization (MALDI-2) provide greatly enhanced sensitivity of detection for a variety of lipids and other small molecules. However, MS imaging of large samples with MALDI-2 at cellular resolution is prohibitively slow for most applications. In this protocol, primary cells are isolated and dispersed onto conductive slides. Relative cell locations are determined by whole-slide fluorescence microscopy, followed by accurate coregistration of the microscopy coordinates to the stage coordinates of the MALDI-2 mass spectrometer. Targeted MS analysis of only cell locations provides high-throughput, single-cell measurements with high analyte coverage and reduced data size as compared to MS imaging of the entire sample. We describe the critical steps necessary for single-cell preparation, whole-slide fluorescence imaging, matrix application, and MALDI-2 mass spectrometry.

This protocol outlines the use of microMS for fluorescence-guided, single-cell MALDI-2 mass spectrometry, enabling enhanced molecular profiling of primary rat neuronal cells.

Single-cell measurements are critical to understanding the rich spatiochemical heterogeneity of the brain. Matrix-assisted laser/desorption ionization ...

Sample Preparation for Rapid Lipid Analysis in Drosophila Brain Using Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging

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