Name: dissection of the drosophila pupal retina for immunohistochemistry, western analysis, and rna isolation
Uploaded: 2019-03-15T15:00:00
Description: Watch this Scientific Journal Video about Dissection of the Drosophila Pupal Retina for Immunohistochemistry, Western Analysis, and RNA Isolation at JoVE.com

We thank Zack Drum and our Reviewers for helpful comments on the manuscript. This work was supported by R15GM114729.&#160;

1. Tissue Preparation
<ol>
	<li>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.</li>
	<li>Maintain Drosophila cultures at 25 &#176;C. For crosses utilizing the UAS-GAL4 system, GMR-GAL4</e...

<ol>
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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...

The Drosophila retina is composed of approximately 750 ommatidia surrounded by pigment cells arranged in a honeycomb lattice1,2,3,4. 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 cell adhesion5,6,7,8,9,10 and apoptosis11,12,13,14,15.&#160;
Several published protocols utilize air pressure to extract eye-brain complexes from Drosophila pupae16,17,18. The protocol described here instead utilizes microdissection tools to carefully and precisely isolate eye-brain complexes with the goal of obtaining undamaged retinal tissue. This is crucial if retinas are to be utilized for morphological, protein, or gene expression analyses since damage to retinas can result in cellular stress or death, which could modify the cellular phenotype or gene expression. Additionally, after practice, 6 to 10 eye-brain complexes can be isolated in 10 to 15 min, facilitating the goal of minimizing variability in the age and developmental stage of dissected eye tissue.
The fixation, immunostaining, and whole-mount protocol described below is suitable for the preparation of Drosophila eyes for fluorescent microscopy. Retinas can be incubated with antibodies targeting proteins of interest. For example, antibodies to adherens junction components can be utilized to visualize the apical circumferences of cells so that characteristics including cell type, shape and arrangement can be assessed19. Prior to fixation, eyes can instead be cleaved from the brain for the purpose of extracting protein for Western analysis, or RNA for use in qRT-PCR or RNA-sequencing.

<table border="0" cellpadding="0" cellspacing="0" style="width:655px;" width="655">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:145px;">Adobe Photoshop</td>
			<td style="width:131px;">Adobe</td>
			<td style="width:95px;"></td>
			<td style="width:285px;">Image processing software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:145px;">Bamboo splints, 6&#34;&#160;</td>
			<td style="width:131px;">Ted Pella Inc</td>
			<td style="width:95px;">116</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:145px;">Beta mercaptoethanol</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:95px;">M3148</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:145px;">Beta-glycerol phosphate</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:95px;">50020</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="90">
			<td height="90" style="height:91px;width:145px;">Black dissecting dish</td>
			<td style="width:131px;"></td>
			<td style="width:95px;"></td>
			<td style="width:285px;">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.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:145px;">Blade holder</td>
			<td style="width:131px;">Fine Science Tools</td>
			<td style="width:95px;">10053</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:145px;">Bovine serum albumin</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:95px;">A7906</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:85px;width:145px;">cOmplete, EDTA-free protease inhibitor cocktail tablets</td>
			<td style="width:131px;">Roche</td>
			<td style="width:95px;">4693132001</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:145px;">Confocal microscope (Zeiss LSM 501)</td>
			<td style="width:131px;">Carl Zeiss</td>
			<td style="width:95px;"></td>
			<td style="width:285px;">or similar microscope</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:145px;">Diethyl Pyrocarbonate (DEPC)</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:95px;">40718</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:145px;">Double-sided tape</td>
			<td style="width:131px;">3M</td>
			<td style="width:95px;">665</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="67">
			<td height="67" style="height:67px;width:145px;">Drosophila food media, nutrient-rich&#160;</td>
			<td style="width:131px;"></td>
			<td style="width:95px;"></td>
			<td style="width:285px;">7.5% sucrose, 15% glucose, 2.5% agar, 20% brewers yeast, 5% peptone, 0.125% MgSO4.7H2O, 0.125% CaCl2.2H20</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:85px;width:145px;">Drosophila food media, standard</td>
			<td style="width:131px;"></td>
			<td style="width:95px;"></td>
			<td style="width:285px;">Bloomington Drosophila Stock center cornmeal recipe.&#160; (https://bdsc.indiana.edu/information/recipes/bloomfood.html)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:145px;">Ethylenediaminetetraacetic acid</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:95px;">E6758</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:145px;">Fixative solution</td>
			<td style="width:131px;"></td>
			<td style="width:95px;"></td>
			<td style="width:285px;">4% formadehyde in PBS, pH 7.4.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:145px;">Fluorescence microscope (TCS SP5 DM microscope)</td>
			<td style="width:131px;">Leica Microsystems</td>
			<td style="width:95px;"></td>
			<td style="width:285px;">or similar microscope</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:145px;">Forceps&#160;</td>
			<td style="width:131px;">Fine Science Tools</td>
			<td style="width:95px;">91150-20</td>
			<td style="width:285px;">Forceps should be sharpened frequently.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:145px;">Formaldehyde</td>
			<td style="width:131px;">Thermo Scientific</td>
			<td style="width:95px;">28908</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:145px;">Glass 9-well dishes&#160;</td>
			<td style="width:131px;">Corning</td>
			<td style="width:95px;">7220-85</td>
			<td style="width:285px;">Also known as 9-well dishes&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:145px;">Glass coverslips (22 x 22 mm)</td>
			<td style="width:131px;">Fisher Scientific</td>
			<td style="width:95px;">12-542-B</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:145px;">Glass microscope slides (25 x 75 x 1 mm)</td>
			<td style="width:131px;">Fisher Scientific</td>
			<td style="width:95px;">12-550-413</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:145px;">Glass petri dish</td>
			<td style="width:131px;">Corning</td>
			<td style="width:95px;">3160-100BO</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:145px;">Glycerol</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:95px;">G5516</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:145px;">Image Studio software version 5.2.5</td>
			<td style="width:131px;">LI-COR Biosciences</td>
			<td style="width:95px;"></td>
			<td style="width:285px;">Image processing software for quantitation of Western blots.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:145px;">Laemmli sample buffer</td>
			<td style="width:131px;">Bio-Rad</td>
			<td style="width:95px;">161-0737</td>
			<td style="width:285px;">2X concentrated protein sample buffer, supplement with beta mercaptoethanol as per manufacturer&#39;s instructions.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:145px;">Lane marker reducing sample buffer&#160;</td>
			<td style="width:131px;">ThermoFisher Scientific</td>
			<td style="width:95px;">39000</td>
			<td style="width:285px;">5X concentrated protein sample buffer.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:145px;">Microcentrigure tubes&#160;</td>
			<td style="width:131px;">Axygen</td>
			<td style="width:95px;">MCT-175-C</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:145px;">Microdissection scissors&#160;</td>
			<td style="width:131px;">Fine Science Tools</td>
			<td style="width:95px;">15000-03</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:145px;">Microwell trays (72 x 10 &#181;L wells)</td>
			<td style="width:131px;">Nunc</td>
			<td style="width:95px;">438733</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:145px;">Mounting media</td>
			<td style="width:131px;"></td>
			<td style="width:95px;"></td>
			<td style="width:285px;">0.5% N-propylgallate and 80% glycerol in PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:145px;">N-propylgallate</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:95px;">P3130</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:145px;">Nuclease-free PBS (PBS in 0.1% DEPC, pH 7.4)</td>
			<td style="width:131px;"></td>
			<td style="width:95px;"></td>
			<td style="width:285px;">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&#176;C</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:145px;">PBS (phosphate buffered saline pH 7.4)</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:95px;">P5368</td>
			<td style="width:285px;">Prepare according to manufacturer&#39;s instructions</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:85px;width:145px;">PBS+pi (PBS plus protease and phoshatase inhibitors)</td>
			<td style="width:131px;"></td>
			<td style="width:95px;"></td>
			<td style="width:285px;">10mM NaF, 1mM beta-glycerol phosphate and 1mM Na3VO4 in PBS, pH 7.4.&#160;&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:145px;">PBT</td>
			<td style="width:131px;"></td>
			<td style="width:95px;"></td>
			<td style="width:285px;">0.15% TritonX and 0.5% bovine serum albumin in PBS, pH 7.4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:145px;">Pin holder</td>
			<td style="width:131px;">Fine Science Tools</td>
			<td style="width:95px;">26016-12</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:145px;">Primary antibody: goat anti-GAPDH</td>
			<td style="width:131px;">Imgenex</td>
			<td style="width:95px;">IMG-3073</td>
			<td style="width:285px;">For Western blotting. Used at 1:3000</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:145px;">Primary antibody: rabbit anti-cleaved Dcp-1</td>
			<td style="width:131px;">Cell signaling</td>
			<td style="width:95px;">9578S</td>
			<td style="width:285px;">For immunofluorescence. Used at 1:100</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:145px;">Primary antibody: rat anti-DEcad</td>
			<td style="width:131px;">Developmental Studies Hybridoma Bank</td>
			<td style="width:95px;">DCAD2</td>
			<td style="width:285px;">For immunofluorescence. Used at 1:20</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:145px;">Primary antibody: rat anti-DEcad</td>
			<td style="width:131px;">DOI: 10.1006/dbio.1994.1287</td>
			<td style="width:95px;">DCAD1</td>
			<td style="width:285px;">&#160;Gift from Tadashi Uemura. Used at 1:100.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:145px;">RNA extration kit: Relia Prep RNA tissue Miniprep kit&#160;</td>
			<td style="width:131px;">Promega</td>
			<td style="width:95px;">Z6110</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:85px;width:145px;">Rnase decontamination reagent (RNase Away)</td>
			<td style="width:131px;">Molecular BioProducts</td>
			<td style="width:95px;">7002</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:145px;">Scalpel blades</td>
			<td style="width:131px;">Fine Science Tools</td>
			<td style="width:95px;">10050</td>
			<td style="width:285px;">Break off small piece of scapel blade and secure in blade holder.</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:85px;width:145px;">Secondary antibody: 488-conjugated&#160; donkey anti-rat IgG (H+L)</td>
			<td style="width:131px;">Jackson ImmunoResearch</td>
			<td style="width:95px;">712-545-153</td>
			<td style="width:285px;">For immunofluorescence. Used at 1:200</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:145px;">Secondary antibody: cy3-conjugated goat anti-rabbit IgG (H+L)</td>
			<td style="width:131px;">Jackson ImmunoResearch</td>
			<td style="width:95px;">111-165-144</td>
			<td style="width:285px;">For immunofluorescence. Used at 1:100</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:85px;width:145px;">Secondary antibody: HRP-conjugated goat anti-rat IgG (H+L)</td>
			<td style="width:131px;">Cell Signaling Technology</td>
			<td style="width:95px;">7077</td>
			<td style="width:285px;">For Western blotting. Used at 1:3000</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:85px;width:145px;">Secondary antibody: HRP-conjugated rabbit anti-goat IgG (H+L)</td>
			<td style="width:131px;">Jackson ImmunoResearch</td>
			<td style="width:95px;">305-035-003</td>
			<td style="width:285px;">For Western blotting. Used at 1:3000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:145px;">Sodium Chloride</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:95px;">S3014</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:145px;">Sodium Fluoride</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:95px;">215309</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:145px;">Sodium vanadate</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:95px;">50860</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:145px;">Spectrophotometer (NanoDrop)</td>
			<td style="width:131px;">ThermoFisher Scientific</td>
			<td style="width:95px;">2000c&#160;</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:145px;">Stereo dissecting microscope (M60 or M80)</td>
			<td style="width:131px;">Leica Microsystems</td>
			<td style="width:95px;"></td>
			<td style="width:285px;">or similar microscope</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:145px;">Sylgard (black)</td>
			<td style="width:131px;">Dow Corning</td>
			<td style="width:95px;">SYLG170</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:145px;">Sylgard (transparent)</td>
			<td style="width:131px;">Dow Corning</td>
			<td style="width:95px;">SYLG184</td>
			<td style="width:285px;">Color black with finely ground charcol powder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:145px;">Tissue: Kimwipes</td>
			<td style="width:131px;">KIMTECH</td>
			<td style="width:95px;">34120</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:145px;">TritonX</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:95px;">T8787</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:145px;">Trizma hydrochloride pH7.5</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:95px;">T5941</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:145px;">Tungsten needle, fine</td>
			<td style="width:131px;">Fine Science Tools</td>
			<td style="width:95px;">10130-10</td>
			<td style="width:285px;">Insert into pin holder</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:145px;">Tungsten needle, sturdy</td>
			<td style="width:131px;">Fine Science Tools</td>
			<td style="width:95px;">10130-20</td>
			<td style="width:285px;">Insert into pin holder</td>
		</tr>
		<tr height="130">
			<td height="130" style="height:130px;width:145px;">WTLB (western tissue lysis buffer)</td>
			<td style="width:131px;"></td>
			<td style="width:95px;"></td>
			<td style="width:285px;">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.</td>
		</tr>
		<tr height="67">
			<td height="67" style="height:67px;width:145px;">Yeast paste</td>
			<td style="width:131px;">(local supermarket)</td>
			<td style="width:95px;"></td>
			<td style="width:285px;">Approximately 2 tablespoons Fleischmann&#39;s ActiveDry Yeast (or similar) dissolved in ~20 mL distilled H2O</td>
		</tr>
	</tbody>
</table>

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.

The pupal eye is an easily-accessible tissue that serves as an excellent model to investigate developmental processes that drive morphogenesis. Here, we have dissected retinas and used immunofluorescence to detect the apical adherens junctions (Figure 3A, C) or the Dcp-1&#160;caspase (Figure 3D) that is activated during apoptosis (Figure 3)25. These approaches allow one to clearly observe cel...

Watch this Scientific Journal Video about Dissection of the Drosophila Pupal Retina for Immunohistochemistry, Western Analysis, and RNA Isolation at JoVE.com

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;

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 ...

This protocol is significant because this is an efficient method for the isolation of Drosophila pupal eye tissue for multiple downstream applications. The main advantage of this technique is that it allows the quick dissection of a large number of pupal eyes without damaging the tissue. Dissection of the Drosophila pupal retina will be challenging for novices.Practicing the technique is the best way to improve the speed of the dissections and the quality of results. After placing the Drosophila pupae onto a black dissecting dish, lay a fresh piece of double-sided tape onto the dissecting dish, away from the pupae, and use a pair of forceps to carefully place the pupae dorsal side up onto the tape. Place the dish under a stereo microscope, and use forceps to remove the operculum of each pupa.Use microdissection scissors to slice each pupal case, allowing it to be flapped open to reveal the head, thorax, and anterior abdominal segment, and secure the edges of the pupal case to the double-sided tape. Pierce the abdomen of each pupa with sharp forceps, and remove the pupa from its pupal case. Place the pupa on the dissection dish, away from the tape, and cover the pupa with 400 microliters of ice-cold PBS.Use forceps to grasp each one by the abdomen, and use the microdissection scissors to make one clean cross-sectional incision through the thorax, cutting the pupae in half. Using two pairs of fine forceps, grasp the cut edges of each thorax epithelium and gradually tear open the thorax and head capsule, exposing the eye-brain complex and the surrounding fat tissue. Then, without grasping the tissue, use forceps to guide each translucent, off-white, dumbbell-shaped eye-brain complex, away from the remnants of the head capsule.After dissecting six to 10 pupae, cut a micropipette tip with a clean razor blade to a diameter of about one millimeter, and lubricate the tip with a mixture of PBS and fat. Use the lubricated pipette tip to transfer the eye-brain complexes to one well of a nine-well glass dish containing 400 microliters of PBS on ice, and then transfer the tissue in 20 microliters of PBS into at least 250 microliters of fixative for a 35-minute incubation on ice. At the end of the fixation, use the same pipette tip to transfer the tissue into a third well containing 400 microliters of fresh PBS for five to 10 minutes on ice.To block any non-specific antibody binding, transfer the eye-brain complexes into 400 microliters of PBS plus Triton X for a 10-to 60-minute incubation on ice. At the end of the incubation, aliquot 10 microliters of the primary antibody solution into the necessary number of wells of a 72-well microwell plate, and use a P10 air-displacement micropipette with a modified 0.5-millimeter tip lubricated with PBS plus Triton X to transfer no more than five eye-brain complexes and no more than three microliters of PBS into each well of antibody solution. After an overnight incubation at four degrees Celsius, wash the samples two times in 400 microliters of PBS plus Triton X in one well of a nine-well glass dish for five to 10 minutes per wash.After the third wash, transfer the tissue into 100 microliters of an appropriate secondary antibody solution for two hours at room temperature, protected from light, following by three five-to 10-minute washes in 400 microliters of PBS plus Triton X and one wash in PBS. Then, equilibrate the samples in 200 microliters of mounting medium for one to two hours. At the end of the incubation, transfer the samples to a microscope slide, and use a sturdy tungsten needle to pin an eye-brain complex to the slide under a dissecting microscope.Then, use a fine tungsten needle to slice each eye away from its associated optic lobe and to maneuver each eye to the surface of the microscope slide so that the basal surface of the eye is adjacent to the slide. Then, apply a clean coverslip over the samples, and seal the coverslip with nail polish. To prepare pupal eye tissue for Western blot analysis, first dissect 10 to 16 pupae as previously demonstrated in two batches, 10 to 15 minutes apart in PBS supplemented with protease and phosphatase inhibitors.After briefly rinsing the isolated eye-brain complexes two times in individual wells of a nine-well glass dish on ice in 400 microliters of PBS plus inhibitors, transfer all of the eye-brain complexes into 400 microliters of ice-cold PBS plus inhibitors decanted onto a clean black dissecting dish. Under a clean dissecting microscope, use a sturdy tungsten needle and a fine razor blade to cleanly cut each eye from the optic lobe, and use a lubricated tip to transfer the eye-brain complexes into five microliters of PBS plus inhibitors into a 500-microliter microcentrifuge tube on ice. Then, add five microliters of Western tissue lysis buffer and 10 microliters of 2X concentrated protein sample buffer to the tube.To process the eye-brain complexes for RNA extraction, wearing gloves, clean the benchtop, microscope, dissecting tools, and black dissecting dishes with 70%ethanol and RNase decontamination reagent. After allowing the area to dry, dissect a total of 30 to 40 pupae in batches of six to eight. After each batch of eye-brain complexes have been dissected, transfer the eye-brain complexes from the nine-well dish into 400 microliters of fresh, ice-cold, nuclease-free PBS on a clean dissecting dish, and use a sturdy tungsten needle and a fine razor blade to cleanly cut each eye from the optic lobe.When all of the eyes for a batch have been removed, transfer them into a sterile, RNase-free microcentrifuge tube, and flash-freeze the samples in liquid nitrogen before proceeding to the next batch. The pupal eye is an easily accessible tissue that serves as an excellent model for investigating developmental processes, including those that drive morphogenesis. Around 40 hours after puparium formation, the final arrangement of cells is achieved.This is an ideal age at which to assess the consequences of a genetic mutation or modified gene expression. For example, following ectopic expression of Diap1, a core inhibitor of apoptosis, the consequent increase in the number of lattice cells can be compared to that observed in a control retina. Apoptosis can also be assessed more directly by utilizing antibodies to activate a death caspase-1 or by using other approaches for detecting dying cells.Western blot analyses of eye lysates can be used to determine the presence or relative expression of proteins of interest or to compare protein expression at different developmental time points. It is important to note that dissecting more than 60 eyes per genotype or experimental condition is optimal for isolating sufficient high-quality RNA. The most important thing to remember is that the pupal retina is extremely fragile, and great care must be taken during its dissection to ensure its integrity.

dissection-drosophila-pupal-retina-for-immunohistochemistry-western

Research

JoVE Journal

Developmental Biology

Live-imaging of the Drosophila Pupal Eye

Inherent processes of Drosophila pupal development can shift and distort the eye epithelium in ways that make individual cell behavior difficult to track during live cell imaging. These processes include: retinal rotation, cell growth and organismal movement. Additionally, irregularities in the topology of the epithelium, including subtle bumps and folds often introduced as the pupa is prepared for imaging, make it challenging to acquire in-focus images of more than a few ommatidia in a single focal plane. The workflow outlined here remedies these issues, allowing easy analysis of cellular processes during Drosophila pupal eye development. Appropriately-staged pupae are arranged in an imaging rig that can be easily assembled in most laboratories. Ubiquitin-DE-Cadherin:GFP and GMR-GAL4&#8211;driven UAS-&#945;-catenin:GFP are used to visualize cell boundaries in the eye epithelium 1-3. After deconvolution is applied to fluorescent images captured at multiple focal planes, maximum projection images are generated for each time point and enhanced using image editing software. Alignment algorithms are used to quickly stabilize superfluous motion, making individual cell behavior easier to track.

Live-imaging of the Drosophila Pupal Eye

This protocol presents an efficient method for imaging the live Drosophila pupal eye neuroepithelium. This method compensates for tissue movement and uneven topology, enhances visualization of cell boundaries through the use of multiple GFP-tagged junction proteins, and uses an easily-assembled imaging rig.

Inherent processes of Drosophila pupal development can shift and distort the eye epithelium in ways that make individual cell behavior difficult to track ...

Isolation of Neural Stem/Progenitor Cells from the Periventricular Region of the Adult Rat and Human Spinal Cord

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of multipotent, self-renewing, and expandable neural stem cells. In serum conditions, these multipotent NSPCs will differentiate, generating neurons, astrocytes, and oligodendrocytes. The harvested tissue is enzymatically dissociated in a papain-EDTA solution and then mechanically dissociated and separated through a discontinuous density gradient to yield a single cell suspension which is plated in neurobasal medium supplemented with epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and heparin. Adult rat spinal cord NSPCs are cultured as free-floating neurospheres and adult human spinal cord NSPCs are grown as adherent cultures. Under these conditions, adult spinal cord NSPCs proliferate, express markers of precursor cells, and can be continuously expanded upon passage. These cells can be studied in vitro in response to various stimuli, and exogenous factors may be used to promote lineage restriction to examine neural stem cell differentiation. Multipotent NSPCs or their progeny can also be transplanted into various animal models to assess regenerative repair.

The adult mammalian spinal cord contains neural stem/progenitor cells (NSPCs) that can be isolated and expanded in culture. This protocol describes the harvesting, isolation, culture, and passaging of NSPCs generated from the periventricular region of the adult spinal cord from the rat and from human organ transplant donors.

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of ...

A Rapid and Efficient Method to Dissect Pupal Wings of Drosophila Suitable for Immunodetections or PCR Assays

Wing development in Drosophila melanogaster is an ideal model for studying morphogenesis at tissue level. These appendages develop from a group of cells named wing imaginal discs formed during embryonic development. In the larval stages the imaginal discs grow, increasing its number of cells and forming monolayered epithelial&#160;structures. Inside the pupal case, the imaginal discs bud out and fold into bilayers along a line that becomes the future margin of the wing. During this process, the longitudinal primodia veins originate vein cells on the prospective dorsal and ventral surfaces of the wing. During the pupal stage the stripes of vein cells of each surface communicate in order to generate tight tubes; at the same time, the cross-veins begin their formation.
With the help of appropriate molecular markers, it is possible to identify the major elements composing the wing during its development. For this reason, the ability to accurately detect transcripts or proteins in this structure is critical for studying their abundance and localization related to the development process of the wing.
The procedure described here focuses on manipulating pupal wings, providing detailed instructions on how to dissect the wing during the pupal stage. The dissection of pupal tissue is more difficult to perform than their counterparts in third instar larvae. This is why this approach was developed, to obtain rapid and efficient high quality samples. Details of how to immunostain and mount these wing samples, to allow the visualization of proteins or cell components, are provided in the protocol. With little expertise it is possible to collect 8-10 high quality pupal wings in a short amount of time.

A Rapid and Efficient Method to Dissect Pupal Wings of Drosophila Suitable for Immunodetections or PCR Assays

The ability to accurately detect transcripts or proteins in Drosophila tissues is critical for studying their abundance and localization related to the development process. Here is the description of a straightforward procedure to dissect pupal wings. These wings can be used as samples in several techniques (immunohistochemistry, PCR assay, etc.).

Wing development in Drosophila melanogaster is an ideal model for studying morphogenesis at tissue level. These appendages develop from a group of cells ...

Correlative Super-resolution and Electron Microscopy to Resolve Protein Localization in Zebrafish Retina

We present a method to investigate the subcellular protein localization in the larval zebrafish retina by combining super-resolution light microscopy and scanning electron microscopy. The sub-diffraction limit resolution capabilities of super-resolution light microscopes allow improving the accuracy of the correlated data. Briefly, 110 nanometer thick cryo-sections are transferred to a silicon wafer and, after immunofluorescence staining, are imaged by super-resolution light microscopy. Subsequently, the sections are preserved in methylcellulose and platinum shadowed prior to imaging in a scanning electron microscope (SEM). The images from these two microscopy modalities are easily merged using tissue landmarks with open source software. Here we describe the adapted method for the larval zebrafish retina. However, this method is also applicable to other types of tissues and organisms. We demonstrate that the complementary information obtained by this correlation is able to resolve the expression of mitochondrial proteins in relation with the membranes and cristae of mitochondria as well as to other compartments of the cell.

This protocol describes the necessary steps to obtain subcellular protein localization results on zebrafish retina by correlating super-resolution light microscopy and scanning electron microscopy images.

We present a method to investigate the subcellular protein localization in the larval zebrafish retina by combining super-resolution light microscopy and ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Dissection and Staining of Drosophila Pupal Ovaries

Unlike adult Drosophila ovaries, pupal ovaries are relatively difficult to access and examine due to their small size, translucence, and encasing within a pupal case. The challenge of dissecting pupal ovaries also lies in their physical location within the pupa: the ovaries are surrounded by fat body cells inside the pupal abdomen, and these fat cells must be removed to allow for proper antibody staining. To overcome these challenges, this protocol utilizes customized Pasteur pipets to extract fat body cells from the pupal abdomen. Moreover, a chambered coverglass is used in place of a microcentrifuge tube during the staining process to improve visibility of the pupae. However, despite these and other advantages of the tools used in this protocol, successful execution of these techniques may still involve several days of practice due to the small size of pupal ovaries. The techniques outlined in this protocol could be applied to time course experiments in which ovaries are analyzed at various stages of pupal development.

Dissection and Staining of Drosophila Pupal Ovaries

The Drosophila ovary is an excellent model system for studying stem cell niche development. Though methods for dissecting larval and adult ovaries have been published, pupal ovary dissections require different techniques that have not been published in detail. Here we outline a protocol for dissecting, staining, and mounting pupal ovaries.

Unlike adult Drosophila ovaries, pupal ovaries are relatively difficult to access and examine due to their small size, translucence, and encasing within a ...

Thawing, Culturing, and Cryopreserving Drosophila Cell Lines

There are currently over 160 distinct Drosophila cell lines distributed by the Drosophila Genomics Resource Center (DGRC). With genome engineering, the number of novel cell lines is expected to increase. The DGRC aims to familiarize researchers with using Drosophila cell lines as an experimental tool to complement and drive their research agenda. Procedures for working with a variety of Drosophila cell lines with distinct characteristics are provided, including protocols for thawing, culturing, and cryopreserving cell lines. Importantly, this publication demonstrates the best practices required to work with Drosophila cell lines to minimize the risk of contaminations from adventitious microorganisms or from other cell lines. Researchers who become familiar with these procedures will be able to delve into the many applications that use Drosophila cultured cells including biochemistry, cell biology and functional genomics.

Thawing, Culturing, and Cryopreserving Drosophila Cell Lines

Drosophila cell lines are important reagents for both fundamental and biomedical research. This article provides protocols for thawing, subculturing, and the cryopreservation of commonly used Drosophila cell lines to assist researchers in incorporating the use of these reagents in their research.

There are currently over 160 distinct Drosophila cell lines distributed by the Drosophila Genomics Resource Center (DGRC). With genome engineering, the ...

Imaging and Analysis of Tissue Orientation and Growth Dynamics in the Developing Drosophila Epithelia During Pupal Stages

Within multicellular organisms, mature tissues and organs display high degrees of order in the spatial arrangements of their constituent cells. A remarkable example is given by sensory epithelia, where cells of the same or distinct identities are brought together via cell-cell adhesion showing highly organized planar patterns. Cells align to one another in the same direction and display equivalent polarity over large distances. This organization of the mature epithelia is established over the course of morphogenesis. To understand how the planar arrangement of the mature epithelia is achieved, it is crucial to track cell orientation and growth dynamics with high spatiotemporal fidelity during development in vivo. Robust analytical tools are also essential to identify and characterize local-to-global transitions. The Drosophila pupa is an ideal system to evaluate oriented cell shape changes underlying epithelial morphogenesis. The pupal developing epithelium constitutes the external surface of the immobile body, allowing long-term imaging of intact animals. The protocol described here is designed to image and analyze cell behaviors at both global and local levels in the pupal abdominal epidermis as it grows. The methodology described can be easily adapted to the imaging of cell behaviors at other developmental stages, tissues, subcellular structures, or model organisms.

Imaging and Analysis of Tissue Orientation and Growth Dynamics in the Developing Drosophila Epithelia During Pupal Stages

This protocol is designed for the imaging and analysis of the dynamics of cell orientation and tissue growth in the Drosophila abdominal epithelia as the fruit fly undergoes metamorphosis. The methodology described here can be applied to the study of different developmental stages, tissues, and subcellular structures in Drosophila or other model organisms.

Within multicellular organisms, mature tissues and organs display high degrees of order in the spatial arrangements of their constituent cells. A ...

Preparation of Drosophila Larval and Pupal Testes for Analysis of Cell Division in Live, Intact Tissue

Experimental analysis of cells dividing in living, intact tissues and organs is essential to our understanding of how cell division integrates with development, tissue homeostasis, and disease processes. Drosophila spermatocytes undergoing meiosis are ideal for this analysis because (1) whole Drosophila testes containing spermatocytes are relatively easy to prepare for microscopy, (2) the spermatocytes&#8217; large size makes them well suited for high resolution imaging, and (3) powerful Drosophila genetic tools can be integrated with in vivo analysis. Here, we present a readily accessible protocol for the preparation of whole testes from Drosophila third instar larvae and early pupae. We describe how to identify meiotic spermatocytes in prepared whole testes and how to image them live by time-lapse microscopy. Protocols for fixation and immunostaining whole testes are also provided. The use of larval testes has several advantages over available protocols that use adult testes for spermatocyte analysis. Most importantly, larval testes are smaller and less crowded with cells than adult testes, and this greatly facilitates high resolution imaging of spermatocytes. To demonstrate these advantages and the applications of the protocols, we present results showing the redistribution of the endoplasmic reticulum with respect to spindle microtubules during cell division in a single spermatocyte imaged by time-lapse confocal microscopy. The protocols can be combined with expression of any number of fluorescently tagged proteins or organelle markers, as well as gene mutations and other genetic tools, making this approach especially powerful for analysis of cell division mechanisms in the physiological context of whole tissues and organs.

Preparation of Drosophila Larval and Pupal Testes for Analysis of Cell Division in Live, Intact Tissue

The goal of this protocol is to analyze cell division in intact tissue by live and fixed cell microscopy using Drosophila meiotic spermatocytes. The protocol demonstrates how to isolate whole, intact testes from Drosophila larvae and early pupae, and how to process and mount them for microscopy.

Experimental analysis of cells dividing in living, intact tissues and organs is essential to our understanding of how cell division integrates with ...

Dissection, Immunohistochemistry and Mounting of Larval and Adult Drosophila Brains for Optic Lobe Visualization

The Drosophila optic lobe, comprised of four neuropils: the lamina, medulla, lobula and lobula plate, is an excellent model system for exploring the developmental mechanisms that generate neural diversity and drive circuit assembly. Given its complex three-dimensional organization, analysis of the optic lobe requires that one understand how its adult neuropils and larval progenitors are positioned relative to each other and the central brain. Here, we describe a protocol for the dissection, immunostaining and mounting of larval and adult brains for optic lobe imaging. Special emphasis is placed on the relationship between mounting orientation and the spatial organization of the optic lobe. We describe three mounting strategies in the larva (anterior, posterior and lateral) and three in the adult (anterior, posterior and horizontal), each of which provide an ideal imaging angle for a distinct optic lobe structure.

Dissection, Immunohistochemistry and Mounting of Larval and Adult Drosophila Brains for Optic Lobe Visualization

This protocol describes three steps to prepare larval and adult Drosophila optic lobes for imaging: 1) brain dissections, 2) immunohistochemistry and 3) mounting. Emphasis is placed on step 3, as distinct mounting orientations are required to visualize specific optic lobe structures.&#160;

The Drosophila optic lobe, comprised of four neuropils: the lamina, medulla, lobula and lobula plate, is an excellent model system for exploring the ...