Name: dissection, immunohistochemistry and mounting of larval and adult drosophila brains for optic lobe visualization
Uploaded: 2021-04-28T14:00:00
Description: Watch this Scientific Journal Video about Dissection, Immunohistochemistry and Mounting of Larval and Adult Drosophila Brains for Optic Lobe Visualization at JoVE.com

We would like to thank Claude Desplan for sharing with us an aliquot of the Bsh antibody. The DE-Cadherin, Dachshund, Eyes Absent, Seven-up and Bruchpilot monoclonal antibodies were obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at The University of Iowa, Department of Biology, Iowa City, IA 52242. This work was supported by an NSERC Discovery Grant awarded to T.E.. U.A. is supported by an NSERC Alexander Graham Bell Canada Graduate Scholarship. P.V. is supported by an Ontario Graduate Scholarship.

1. Preparing larval brains for confocal imaging
<ol>
	<li>Dissections 
	NOTE: Before starting the dissection, prepare the fix (4% formaldehyde in phosphate buffered saline (PBS)) and PBT (0.1-0.3% Triton in PBS) solutions. The fix solution should be placed on ice during the dissection. Although paraformaldehyde (PFA) fixative is used in this protocol, alternative fixing strategies (using PLP20 or PEM21) have been described for specific epitopes. For ...

<ol>
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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+Principles+of+Insect+and+Vertebrate+Visual+Systems.">Design Principles of Insect and Vertebrate Visual Systems.</a> Neuron. 66, 15-36 (2010).</li><li>N&#233;riec, N., Desplan, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+the+Eye+to+the+Brain.+Development+of+the+Drosophila+Visual+System.">From the Eye to the Brain. Development of the Drosophila Visual System.</a> Current Topics in Developmental Biology. 116, 247-271 (2016).</li><li>Apitz, H., Salecker, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Challenge+of+Numbers+and+Diversity:+Neurogenesis+in+the+Drosophila+Optic+Lobe.">A Challenge of Numbers and Diversity: Neurogenesis in the Drosophila Optic Lobe.</a> Journal of Neurogenetics. 28, 233-249 (2014).</li><li>Contreras, E. G., Sierralta, J., Oliva, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+strategies+for+the+generation+of+neuronal+diversity:+Lessons+from+the+fly+visual+system.">Novel strategies for the generation of neuronal diversity: Lessons from the fly visual system.</a> Frontiers in Molecular Neuroscience. 12, 140 (2019).</li><li>Erclik, T., Hartenstein, V., Lipshitz, H. D., McInnes, R. 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In this protocol, we describe a method to immunostain larval and adult Drosophila brains and mount them in several orientations. While methods to stain larval and adult brains have been previously described22,23,24,27,28, mounting strategies for the optimal visualization of specific optic lobe structures have received less attention2...

The Drosophila visual system, comprised of the compound eye and underlying optic lobe, has been an excellent model for the study of neural circuit development and function. In recent years, the optic lobe in particular has emerged as a powerful system in which to study neurodevelopmental processes such as neurogenesis and circuit wiring1,2,3,4,5,6,7,8. It is made up of four neuropils: the lamina, medulla, lobula and lobula plate (the latter two comprise the lobula complex)1,2,3,4,5,6. Photoreceptors from the eye, target neurons of the lamina and medulla, which process visual inputs and relay them to the neuropils of the lobula complex1,2,3,4,5,6. Projection neurons in the lobula complex subsequently send visual information to the higher order processing centers in the central brain1,5,9. The complex organization of the optic lobe, necessitated by a need to maintain retinotopy and to process different types of visual stimuli, makes it an attractive system for studying how sophisticated neural circuits are assembled. Notably, the medulla shares striking similarities in both its organization and development with the neuroretina, which has long been a model for vertebrate neural circuit development3,8.
Optic lobe development begins during embryogenesis, with the specification of ~35 ectodermal cells that form the optic placode2,4,5,6,7,8. After larval hatching, the optic placode is subdivided into two distinct primordia: 1) the outer proliferation center (OPC), which generates the neurons of the lamina and outer medulla and 2) the inner proliferation center (IPC), which generates neurons of the inner medulla and lobula complex4,5,6,10. In the late second-instar larva, the neuroepithelial cells of the OPC and IPC begin to transform into neuroblasts that subsequently generate neurons via intermediate ganglion mother cells4,5,11,12. Optic lobe neuroblasts are patterned by spatially and temporally-restricted transcription factors, which act together to generate neural diversity in their progeny11,12,13,14. In the pupa, the circuits of the optic lobe neuropils are assembled via the coordination of several processes, including programmed cell death11,15, neuronal migration12,16, axonal/dendritic targeting10,17, synapse formation18,19 and neuropil rotations10,17.
Here, we describe the methodology by which larval and adult brains are dissected, immunostained and mounted for imaging the optic lobe. 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. Thus, we put special emphasis on how the orientation of mounting relates to the spatial organization of the optic lobe structures. We describe three mounting strategies for larval brains (anterior, posterior and lateral) and three for adult brains (anterior, posterior and horizontal), each of which provide an optimal angle for imaging a specific optic lobe progenitor population or neuropil.

<table border="0" cellpadding="0" cellspacing="0" style="width:1077px;" width="1077">
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	<tbody>
		<tr height="26">
			<td height="26" style="height:27px;width:231px;">10x PBS</td>
			<td style="width:325px;">Bioshop</td>
			<td style="width:164px;">PBS405</td>
			<td style="width:357px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:231px;">37% formaldehyde</td>
			<td style="width:325px;">Bioshop</td>
			<td style="width:164px;">FOR201</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:231px;">Alexa Fluor 488 (goat) secondary</td>
			<td style="width:325px;">Invitrogen</td>
			<td style="width:164px;">A-11055</td>
			<td>use at 1:501</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:231px;">Alexa Fluor 555 (mouse) secondary</td>
			<td style="width:325px;">Invitrogen</td>
			<td style="width:164px;">A-31570</td>
			<td>use at 1:500</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:231px;">Alexa Fluor 647 (guinea pig) secondary</td>
			<td style="width:325px;">Invitrogen</td>
			<td style="width:164px;">A-21450</td>
			<td>use at 1:503</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:231px;">Alexa Fluor 647 (rat) secondary</td>
			<td style="width:325px;">Invitrogen</td>
			<td style="width:164px;">A-21247</td>
			<td>use at 1:502</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:231px;">Cover slips</td>
			<td style="width:325px;">VWR</td>
			<td style="width:164px;">48366-067</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:231px;">Dissecting forceps - #5</td>
			<td style="width:325px;">Dumont</td>
			<td style="width:164px;">11251-10</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:231px;">Dissecting forceps - #55</td>
			<td style="width:325px;">Dumont</td>
			<td style="width:164px;">11295-51</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:231px;">Dissection Dish</td>
			<td style="width:325px;">Corning</td>
			<td>722085</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:231px;">Dry wipes</td>
			<td style="width:325px;">Kimbery Clark</td>
			<td style="width:164px;">34155</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:231px;">Goat anti-Bgal primary antibody</td>
			<td>Biogenesis</td>
			<td></td>
			<td>use at 1:1000</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:231px;">Guinea pig anti-Bsh primary antibody</td>
			<td style="width:325px;">Gift from Claude Desplan</td>
			<td style="width:164px;"></td>
			<td style="width:357px;">use at 1:500</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:231px;">Guinea pig anti-Vsx1 primary antibody</td>
			<td style="width:325px;">Erclik et al. 2008</td>
			<td style="width:164px;"></td>
			<td style="width:357px;">use at 1:1000</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:231px;">Laboratory film</td>
			<td style="width:325px;">Parfilm</td>
			<td style="width:164px;">PM-996</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:231px;">Microcentrifuge tubes</td>
			<td style="width:325px;">Sarstedt</td>
			<td style="width:164px;">72.706.600</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:231px;">Microscope slides</td>
			<td style="width:325px;">VWR</td>
			<td style="width:164px;">CA4823-180</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:231px;">Mouse anti-dac primary antibody</td>
			<td style="width:325px;">Developmental Studies Hybridoma Bank (DSHB)</td>
			<td style="width:164px;">mabdac2-3</td>
			<td>use at 1:20</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:231px;">Mouse anti-eya primary antibody</td>
			<td style="width:325px;">DSHB</td>
			<td style="width:164px;">eya10H6</td>
			<td>use at 1:20</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:231px;">Mouse anti-nc82 primary antibody</td>
			<td style="width:325px;">DSHB</td>
			<td style="width:164px;">nc82</td>
			<td>use at 1:50</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:231px;">Mouse anti-svp primary antibody</td>
			<td style="width:325px;">DSHB</td>
			<td style="width:164px;">Seven-up 2D3</td>
			<td>use at 1:100</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:231px;">Polymer Clay</td>
			<td>Any type of clay can be used</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:231px;">Rabbit anti-GFP</td>
			<td style="width:325px;">Invitrogen</td>
			<td style="width:164px;">A-11122</td>
			<td>use at 1:1000</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:231px;">Rat anti-DE-Cadherin primary antibody</td>
			<td style="width:325px;">DSHB</td>
			<td style="width:164px;">DCAD2</td>
			<td>use at 1:20</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:231px;">Slowfade mounting medium</td>
			<td style="width:325px;">Invitrogen</td>
			<td style="width:164px;">S36967</td>
			<td>Vectashield mounting medium ( cat# H-1000) can also be used</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:231px;">Triton-x-100</td>
			<td style="width:325px;">Bioshop</td>
			<td style="width:164px;">TRX506</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Confocal images of larval and adult optic lobes mounted in the orientations described in the protocol are presented in Figure 1 and Figure 2.
Figure 1 shows schematics and representative confocal slices of larval brains positioned in the anterior, posterior and lateral orientations. In the anterior mounting orientation, the OPC epithelium (DE-Cadherin), medulla neuroblasts (deadpan&#62;&#946;gal)...

Watch this Scientific Journal Video about Dissection, Immunohistochemistry and Mounting of Larval and Adult Drosophila Brains for Optic Lobe Visualization at JoVE.com

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;

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 ...

Research

JoVE Journal

Developmental Biology

Dissection of Larval Zebrafish Gonadal Tissue

Although wild zebrafish possess a ZZ/ZW sex-determination system, domesticated zebrafish have lost the sex chromosome. They utilize a polygenic sex ...

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

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 ...

Dissection and Staining of Drosophila Pupal Ovaries

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 ...

Visualization of Tangential Cell Migration in the Developing Chick Optic Tectum

Time-lapse imaging is a powerful method to analyze migrating cell behavior. After fluorescent cell labeling, the movement of the labeled cells in culture ...

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

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 ...

Visualization and Analysis of Pharyngeal Arch Arteries using Whole-mount Immunohistochemistry and 3D Reconstruction

Improper formation or remodeling of the pharyngeal arch arteries (PAAs) 3, 4, and 6 contribute to some of the most severe forms of congenital heart ...

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

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 ...

A Protocol for Immunohistochemistry and RNA In-situ Distribution within Early Drosophila Embryo

A Protocol for Immunohistochemistry and RNA In-situ Distribution within Early Drosophila Embryo

Calcium induced calcium release signaling (CICR) plays a critical role in many biological processes. Every cellular activity from cell proliferation and ...

Dissection and Live-Imaging of the Late Embryonic Drosophila Gonad

Dissection and Live-Imaging of the Late Embryonic Drosophila Gonad

The Drosophila melanogaster male embryonic gonad is an advantageous model to study various aspects of developmental biology including, but not limited to, ...

Stab Wound Injury Model of the Adult Optic Tectum Using Zebrafish and Medaka for the Comparative Analysis of Regenerative Capacity

While zebrafish have a superior capacity to regenerate their central nervous system (CNS), medaka has a lower CNS regenerative capacity. A brain injury ...