Name: confocal and super-resolution imaging of polarized intracellular trafficking and secretion of basement membrane proteins during drosophila oogenesis
Uploaded: 2022-05-19T14:00:00
Description: Watch this Scientific Journal Video about Confocal and Super-Resolution Imaging of Polarized Intracellular Trafficking and Secretion of Basement Membrane Proteins During Drosophila Oogenesis at JoVE.c

The authors are grateful to Julie Merkle for her helpful comments on the manuscript. This work was supported by NIH grant R15GM137236 to O.D. The confocal and super-resolution images were acquired using a Zeiss LSM 900 with Airyscan 2, purchased with NSF MRI grant 2018748.

1. Fly preparation for ovary dissections
<ol>
	<li>Put 10-15 Drosophila melanogaster&#160;female flies (1-2 days old) of the desired genotype in a narrow vial containing ~8 mL of Drosophila fly medium sprinkled with a small amount of granulated baker&#39;s yeast 2-3 days prior to dissection at 25 &#176;C. Adding a few males to the vial can boost egg chamber yield. However, ensure that the total number of flies does not exceed 20 as this can negatively affect ovary development. 
	...

The BM is critical for embryonic and organ morphogenesis, and adult physiological functions. Moreover, the BM acts as a signaling platform for the establishment and maintenance of epithelial polarity and provides tissues with support2. Yet, the mechanisms that regulate the proper placement of BM proteins are poorly understood. A better understanding of the biological pathways dedicated to the intracellular trafficking and polarized secretion of BM proteins requires a careful analysis of the compon...

The basement membrane (BM) is a thin sheet of layered cell-adherent extracellular matrix (ECM) critical for epithelial structure and morphogenesis1. It comprises ~50 proteins and is found ubiquitously underlying the epithelial and endothelial cells, and ensheathing skeletal, smooth, and heart muscle cells and adipocytes1,2,3. The three main components of the BM at the basal side of the epithelial cells are Collagen IV, Perlecan, and Laminins. The BM underlies the epithelial cells and is responsible for many functions, including tissue separation and barrier, growth and support, and cell polarization2,3,4,5,6,7,8,9,10,11,12. Its role as a signaling platform regulates the morphology and differentiation of epithelial cells and tissues during development3,13,14. Moreover, the mis-regulation of the BM and/or a breach in its integrity are the primary causes of many pathological conditions, including tumor metastasis2,15,16. Despite the essential functions performed by the BM during tissue and organ morphogenesis, the components of the biological pathway(s) dedicated to the polarized intracellular trafficking and secretion of BM proteins are vaguely known.
To study the intracellular trafficking of BM-containing vesicles and the secretion of BM proteins by epithelial cells, the follicular epithelium (FE) of the Drosophila ovary is a powerful model system (Figure 1). A Drosophila ovary comprises 16-20 long, tube-like structures, called ovarioles (Figure 1A,B)17,18,19. Each ovariole can be thought of as an egg assembly line, with the age progression of egg chambers (which each gives rise to an egg) that begins at the anterior end and moves posteriorly, until the mature egg exits through the oviduct. Each egg chamber is encapsulated by the FE, a monolayer of somatic follicle cells (FCs), that surrounds the central germline cells (GCs). The FE is highly polarized with a distinct apical-basal polarity where the apical domain faces the germline, and the BM proteins are secreted basally18,19. The FCs actively secrete all of the major components of the BM, including Collagen IV, Perlecan, and Laminins20,21. In epithelial cells such as FCs, the BM components are produced and require a specialized polarized secretion pathway for their deposition extracellularly. For example, in the case of the most abundant component of the BM, Collagen IV (Coll IV), the details surrounding its polarized intracellular trafficking and secretion are vague despite its production and deposition being the focus of many studies. Coll IV is translated in the endoplasmic reticulum (ER), which is also where each fibril - composed of three polypeptides (two &#945;1 chains and one &#945;2 chain) - is assembled into a triple helix22. Proper Coll IV folding and function require ER chaperones and enzymes, including lysyl and prolyl-hydroxylases such as Plod and PH4&#945;EFB20,22,23,24,25,26. These posttranslational enzymes regulate the ER sorting of Coll IV, as the loss of each causes Coll IV to be trapped in the basal ER20,23,24,25,26. Then, newly synthesized Coll IV exits the ER for the Golgi in COPII-coated vesicles. The cargo receptor Tango1 aids in packaging collagens into sizable Golgi-bound vesicles that can accommodate large multimeric proteins20,27. Once Coll IV is packaged into intracellular exocytic vesicles, it is specifically secreted basally from epithelial cells. To direct BM deposition to the basal side, epithelial cells require another set of factors specifically dedicated to polarized BM secretion. Using the FE of the Drosophila ovary, a few components of this novel cellular process have been characterized, including the nucleotide exchange factors (GEFs) Crag and Stratum, the GTPases Rab8 and Rab10, as well as the levels of the phosphoinositide PI(4,5)P2, and Kinesin 1 and 3 motor proteins20,28,29,30,31. These components are critical in ensuring the polarized distribution of BM proteins.
To monitor the intracellular localization of BM proteins in the FE, endogenously tagged basement membrane proteins (protein traps), such as Viking-GFP (Vkg-GFP or &#945;2 Coll IV-GFP) and Perlecan-GFP (Pcan-GFP) can be used32,33. These protein trap lines have been shown to accurately reflect the endogenous distribution of BM proteins and allow for more sensitive detection of vesicular trafficking28,30. The components involved in the polarized deposition of BM in the FE were first characterized using protein trap lines for Vkg-GFP and Pcan-GFP20,28,29,30. Protein traps can be used in different genetic backgrounds, including mutants and Gal4 lines34. Moreover, protein traps can be used in combination with fluorescent dyes and/or fluorescence immunostaining, allowing for precise characterization of the localization of BM proteins when comparing wild-type and mutant conditions35.
To accurately and efficiently assess the distribution and localization of BM protein-containing vesicles, confocal laser scanning microscopy (CLSM) and super-resolution imaging techniques present a significant advantage to other imaging approaches. These approaches couple high-resolution imaging with relative ease of use. CLSM is a microscopy technique that allows for an improved optical resolution by scanning the specimen with a laser in a raster scan manner using galvanometers. The pinhole aperture is a core component of a confocal microscope. By blocking the out-of-focus signals coming from above or below the focal plane, the pinhole aperture results in a highly superior resolution in the z-axis36. This also makes it possible to obtain a series of images in the z-plane, called a z-stack, corresponding to a series of optical sections. z-stacks subsequently create a 3D image of the specimen, via 3D reconstruction, with the aid of imaging software. Conventional epifluorescence (widefield) microscopes, unlike confocal microscopes, allow out-of-focus light to contribute to image quality, decreasing image resolution and contrast36,37. This makes epifluorescence microscopy a less attractive candidate when studying protein localization or colocalization.
Although CLSM is a suitable approach for various applications, including imaging and characterization of the intracellular trafficking of BM proteins, it still presents an issue when imaging samples below Abbe&#39;s diffraction limit of light (200-250 nm). When imaging such samples, confocal microscopy, especially when using an oil objective, can result in high resolution. However, super-resolution techniques surpass the limit of confocal microscopy. There are various approaches to achieve super-resolution microscopy, each with specific resolution limits, and each appropriate for different analyses. These approaches include photoactivated localization microscopy (PALM) or stochastic optical reconstruction microscopy (STORM), stimulated emission depletion microscopy (STED), structured illumination microscopy (SIM), and Airyscan (super-resolution) microscopy38,39,40,41,42,43,44,45,46. Although Airyscan has a coarser resolution than PALM/STORM, STED, and SIM, it can still achieve a resolution of up to ~120 nm (about twice the resolution of CLSM). Furthermore, this super-resolution microscopy approach has been shown to have an advantage over SIM and other super-resolution techniques when imaging thick samples and samples with a low signal-to-noise ratio47,48.
Airyscan is a relatively new super-resolution confocal microscopy technology46. Unlike traditional CLSMs, which use the pinhole and single point detectors to reject out-of-focus light, this super-resolution approach uses a 32-channel gallium arsenide phosphide (GaAsP) photomultiplier tube area detector that collects all of the light at every scan position45. Each of the 32 detectors work as a small pinhole, reducing the pinhole size from the traditional 1.0 Airy Unit (A.U.) to an enhanced 0.2 A.U., enabling an even higher resolution and signal-to-noise ratio, while maintaining the efficiency of a 1.25 A.U. diameter45. Furthermore, the linear deconvolution used by Airyscan results in up to a 2x increase in resolution45. Taking this into consideration, CLSM, and specifically super-resolution microscopy, are&#160;well-suited to study BM proteins and proteins that regulate the basal deposition of BM proteins, as they can produce very high-resolution images for localization and colocalization studies, thereby providing new insights in the spatial, temporal, and molecular events that control these processes.
An alternative approach to confocal microscopy that can be used to perform localization experiments is image deconvolution. Since widefield microscopy allows out-of-focus light to reach the detectors, mathematical and computational deconvolution algorithms can be applied to remove or reassign out-of-focus light from images obtained by widefield microscopy, thereby improving the resolution and contrast of the image49. Deconvolution algorithms can also be applied to confocal images to further increase resolution and contrast, producing final images almost comparable to that of super-resolution microscopy50. Airyscan makes use of Weiner filter-based deconvolution along with Sheppard&#39;s pixel reassignment, resulting in a highly improved spatial resolution and signal-to-noise ratio. Compared to confocal microscopy, an increase of 2x in resolution in all three spatial dimensions (120 nm in x and y, and 350 nm in z) is observed when using this super-resolution microscopy technique45,51.
This manuscript provides detailed and optimized protocols to stain, acquire, and visualize the intracellular trafficking and deposition of BM proteins using the FE of the Drosophila ovary as a model system coupled with confocal and super-resolution microscopy. Drosophila lines expressing endogenously tagged basement membrane proteins, e.g., Vkg-GFP and Pcan-GFP, are efficient and accurate tools to visualize BM protein trafficking and secretion. In addition, they can be easily used in different genetic backgrounds, including mutant and Gal4/UAS lines34. Although endogenously tagged basement membrane proteins are recommended, the use of antibodies against specific BM proteins is also compatible with the described protocols. These protocols are particularly useful for scientists who are interested in studying intracellular trafficking and the secretion of BM proteins in intact epithelial tissue using confocal and super-resolution imaging. Moreover, the ability to combine epithelial tissue analysis with the expansive tools of Drosophila genetics makes this approach especially powerful. Finally, these protocols could be easily adapted to study vesicular trafficking and sorting of other proteins of interest.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Alexa Fluor 546 phalloidin</td>
			<td>Invitrogen</td>
			<td>A22283</td>
			<td>F-Actin Stain (1/500 of 66&#181;M)</td>
		</tr>
		<tr>
			<td>Alexa Fluor 647 phalloidin</td>
			<td>Invitrogen</td>
			<td>A22287</td>
			<td>F-Actin Stain (1/100 of 66&#181;M))</td>
		</tr>
		<tr>
			<td>Anti-GM130 Antibody</td>
			<td>abcam</td>
			<td>ab30637</td>
			<td>For Golgi Stain (colocalization); use as concentration of 7&#181;g/uL</td>
		</tr>
		<tr>
			<td>Aqua-Polymount</td>
			<td>Polysciences, Inc.</td>
			<td>1860620</td>
			<td>Mounting Medium</td>
		</tr>
		<tr>
			<td>Bakers Yeast (Active Dry Yeast)</td>
			<td>Genesee Scientific</td>
			<td>62-103</td>
			<td>To fatten the overies for dissection</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (30% solution)</td>
			<td>Sigma-Aldrich</td>
			<td>A7284</td>
			<td>For blocking solution</td>
		</tr>
		<tr>
			<td>Depression wells</td>
			<td>Electron Microscopy Sciences</td>
			<td>7156101</td>
			<td>For dissection (glass concavity slide can be used instead)</td>
		</tr>
		<tr>
			<td>Dissecting needle</td>
			<td>Fisher scientifc</td>
			<td>13-820-024</td>
			<td></td>
		</tr>
		<tr>
			<td>Drosophila Incubator</td>
			<td>Genesee Scientific/Invictus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fly Stock: Perlecan-GFP Drosophila line (ZCL1700)</td>
			<td></td>
			<td></td>
			<td>Morin et al., 2001</td>
		</tr>
		<tr>
			<td>Fly Stock: UAS-Crag RNAi line (TRIP line HMS00241)</td>
			<td>Bloomington Drosophila Stock Center</td>
			<td>33594</td>
			<td>RNAi against Crag</td>
		</tr>
		<tr>
			<td>Fly Stock: Viking-GFP Drosophila line (CC00791)</td>
			<td></td>
			<td></td>
			<td>Buszczak et al., 2007</td>
		</tr>
		<tr>
			<td>Fly Stock: Vkg-GFP, tj-Gal4</td>
			<td></td>
			<td></td>
			<td>Devergne et al., 2017. Drive the expression of Crag RNAi in the FE</td>
		</tr>
		<tr>
			<td>Forceps (Dumont 5)</td>
			<td>Fine Science Tools</td>
			<td>11251-30</td>
			<td>For dissection</td>
		</tr>
		<tr>
			<td>Glass Concavity Slide</td>
			<td>Electron Microscopy Sciences</td>
			<td>7187804</td>
			<td>For dissection (depression wells can be used instead)</td>
		</tr>
		<tr>
			<td>Goat anti-Rabbit IgG, Alexa Fluor 568</td>
			<td>Invitrogen</td>
			<td>A11036</td>
			<td>Secondary antibody (GM130 antibody) (5 &#181;g/mL)</td>
		</tr>
		<tr>
			<td>Hoechst (Hoechst 33342)</td>
			<td>Invitrogen</td>
			<td>H3570</td>
			<td>DNA Stain (1 ug/mL)</td>
		</tr>
		<tr>
			<td>Kimwipes</td>
			<td>Kimtech</td>
			<td>Fisher Scientific: 06-666</td>
			<td>Delicate task wipers</td>
		</tr>
		<tr>
			<td>Leica Fluorescent Stereo Microscope&#160; M165 FC</td>
			<td>Leica</td>
			<td></td>
			<td>For ovary imaging</td>
		</tr>
		<tr>
			<td>Microscope Slides</td>
			<td>Corning</td>
			<td>294875X25</td>
			<td>Microscope Slides</td>
		</tr>
		<tr>
			<td>Nutating platform rocker</td>
			<td>Corning Life Sciences</td>
			<td>6720</td>
			<td>For ovary fixation and staining</td>
		</tr>
		<tr>
			<td>Nutri-Fly BF</td>
			<td>Genesee Scientific</td>
			<td>66-121</td>
			<td>Fly Food</td>
		</tr>
		<tr>
			<td>Paraformaldehyde 20% Solution</td>
			<td>Electron Microscopy Sciences</td>
			<td>Fisher Scientific: 15713</td>
			<td>For PFA 4%</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline Tablets</td>
			<td>Fisher scientific</td>
			<td>BP2944100</td>
			<td>For PBS solution</td>
		</tr>
		<tr>
			<td>ProLong Glass Antifade Mountant</td>
			<td>Invitrogen</td>
			<td>P36980</td>
			<td>Mounting Medium</td>
		</tr>
		<tr>
			<td>Square Cover Glass</td>
			<td>Corning</td>
			<td>285022</td>
			<td>Cover glass for microscope slides</td>
		</tr>
		<tr>
			<td>Triton x-100</td>
			<td>Sigma-Aldrich</td>
			<td>9036-19-5</td>
			<td>For PBST</td>
		</tr>
		<tr>
			<td>Zeiss LSM 900 with Airyscan 2</td>
			<td>Zeiss</td>
			<td></td>
			<td>Confocal and super-resolution Microscope</td>
		</tr>
		<tr>
			<td>Zeiss Stemi 305 Stereo Microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td>Dissecting microscope</td>
		</tr>
		<tr>
			<td>Zeiss Zen Software version 3.3 (Blue Edition)</td>
			<td>Zeiss</td>
			<td></td>
			<td>Image acquisition and processing</td>
		</tr>
	</tbody>
</table>

confocal and super-resolution imaging of polarized intracellular trafficking and secretion of basement membrane proteins during drosophila oogenesis

The basement membrane (BM) - a specialized sheet of extracellular matrix present at the basal side of epithelial cells - is critical for the establishment and maintenance of epithelial tissue morphology and organ morphogenesis. Moreover, the BM is essential for tissue modeling, serving as a signaling platform, and providing external forces to shape tissues and organs. Despite the many important roles that the BM plays during normal development and pathological conditions, the biological pathways controlling the intracellular trafficking of BM-containing vesicles and how basal secretion leads to the polarized deposition of BM proteins are poorly understood. The follicular epithelium of the Drosophila ovary is an excellent model system to study the basal deposition of BM membrane proteins, as it produces and secretes all major components of the BM. Confocal and super-resolution imaging combined with image processing in fixed tissues allows for the identification and characterization of cellular factors specifically involved in the intracellular trafficking and deposition of BM proteins. This article presents a detailed protocol for staining and imaging BM-containing vesicles and deposited BM using endogenously tagged proteins in the follicular epithelium of the Drosophila ovary. This protocol can be applied to address both qualitative and quantitative questions and it was developed to accommodate high-throughput screening, allowing for the rapid and efficient identification of factors involved in the polarized intracellular trafficking and secretion of vesicles during epithelial tissue development.

The methods described herein can be used to efficiently and accurately image and characterize the intracellular trafficking and secretion of BM proteins in polarized epithelial cells, such as the FE of the Drosophila ovary. Next, we provide anticipated results obtained using the described methods, as well as helpful advice and potential pitfalls. To do so, Vkg-GFP, an endogenously tagged Vkg (Drosophila Col IV) is used. However, the same results can be achieved with other endogenously tagged BM proteins...

Watch this Scientific Journal Video about Confocal and Super-Resolution Imaging of Polarized Intracellular Trafficking and Secretion of Basement Membrane Proteins During Drosophila Oogenesis at JoVE.c

Confocal and Super-Resolution Imaging of Polarized Intracellular Trafficking and Secretion of Basement Membrane Proteins During Drosophila Oogenesis

The basement membrane is essential for tissue and organ morphogenesis during development. To better understand the mechanisms leading to proper placement of this structure, the protocol presented describes methods to visualize and characterize the intracellular trafficking and secretion of basement membrane proteins in epithelial cells using confocal and super-resolution microscopy.

Confocal and Super-Resolution Imaging of Polarized Intracellular Trafficking and Secretion of Basement Membrane Proteins During Drosophila Oogenesis

The basement membrane (BM) - a specialized sheet of extracellular matrix present at the basal side of epithelial cells - is critical for the establishment ...

This protocol allows the characterization of the intracellular trafficking and secretion of basement membrane proteins using the Drosophila array as a model system. The main advantage of our technique is that it allows high-resolution imaging of the intracellular trafficking of basement membrane proteins using endogenously-tagged proteins and Airyscan super-resolution microscopy. Although this protocol is developed to image the intracellular trafficking of basement membrane proteins, it can be extended to study the trafficking of other proteins of interest and other biological systems, including cell culture and organoids.To begin, after dissecting Drosophila ovaries in PBS under a dissecting scope, add one milliliter of fixation solution and fix the ovaries on a nutating platform for 15 minutes. Remove the fixation solution and perform two quick washes, each with one milliliter of PBST. By gently inverting the microcentrifuge tubes five to six times, then perform four long washes of 10 minutes each with one milliliter of PBST on a nutating platform rocker.After performing fixation and washing, remove PBST, then add one milliliter of blocking solution and block the ovaries on a nutating platform rocker for a minimum of one hour. Next, remove the blocking solution and add 300 microliters of primary antibody solution, containing primary antibodies diluted at their appropriate concentrations in blocking solution. Incubate overnight on a nutating platform rocker at 4 degrees Celsius.After removing the primary antibody solution, perform two quick washes and four long washes, as demonstrated previously, then remove PBST and add 500 microliters of secondary antibody solution containing fluorescent secondary antibodies that will detect the primary antibodies used. Incubate the ovaries in the secondary antibody solution on a nutating platform rocker for two hours at room temperature, then perform two quick washes and four long washes in PBST as demonstrated previously on a nutating platform rocker. After the last wash, use a P-1000 pipette to gently pipette the ovaries up and down in the tube to separate the egg chambers.Allow the egg chambers to sink to the bottom by keeping the tube in an upright position for five to 10 minutes. Next, remove PBST using a Pasteur pipette, leaving approximately 50 microliters. Afterward, remove as much of the remaining PBST as possible using a P-200 pipette and add two drops of mounting medium, enough to spread evenly on a cover slip.To allow easy transfer of the viscous mounting medium to the slide from the microcentrifuge tube, cut off the end of a P-200 pipette tip. Next, slowly transfer all the egg chambers in the mounting medium to the glass slide, ensuring not to create bubbles. Under a dissecting microscope, gently spread out the mounting media and separated egg chambers using a new P-200 pipette tip or forceps to cover an area approximately the size of a cover slip.Using forceps, carefully place the cover slip on the egg chambers at an angle to avoid bubbles and store the slide at room temperature on a flat surface in the dark for two days to polymerize. Once the mounting media has cured, the slide can be stored at 4 degrees Celsius in the dark for a few weeks for imaging. To visualize the intracellular localization of basement membrane proteins, set the objective to 63x and gently place a drop of immersion oil on its lens, then position the slide on the objective with the cover slip facing the objective to locate the specimen.Locate the region of interest using the eyepiece of the epifluorescent microscope, then select a configuration with appropriate settings for the fluorophores to image as described in the manuscript. Once the configuration is set, image the sample by selecting Live under Acquisition Mode and adjust the zoom between 2 and 4x to optimize the scan area of the sample. For each individual channel, select a track under Channel and click on Live.Adjust the master gain and laser power while using the range indicator tool and follow all the guidelines to avoid saturated pixels as described in the manuscript, while repeating the same for each channel. In the Acquisition Mode toggle window, under Image Size, click on SR to maximize the capabilities of the detector and adjust the frame size automatically. Keep the averaging to None in Airyscan mode as it is usually not necessary and will decrease the scan time.However, in some cases, an averaging of 2x may improve the signal-to-noise ratio, then click on Snap to acquire an image. To acquire a Z-stack, click on Z-Stack checkbox under the Acquisition tab. Next, select the desired channel to observe the specimen.For example, the DAPI channel and click on Live to start a live scan, then set a range for the Z-stack using the fine adjustment knob on the microscope. Afterward, set the endpoints of the Z-stack by clicking Set First and Set Last. For optimal 3D reconstruction, set the interval for the Z-stack lower than 0.5 micrometers to assign the step size and click on Start Experiment to begin Z-stack acquisition.Once the images or Z-stack is obtained, click on the Processing, then Method option and select Airyscan Processing. Perform Auto Filter to start and if required, perform further manual processing by changing the super resolution value to obtain the best results for the sample. After the optimal SR value has been determined, click on Apply to generate a processed image.In the case of Z-stack images, process either as one Z-slice or as the whole Z-stack by clicking on the 3D Processing box. To visualize protein trafficking in 3D after the acquisition of a Z-stack, generate a 3D image by clicking on the 3D icon in the preview window, that will appear in the display control section of the Airyscan processed image. From the different 3D view options, use Surface or Mixed views when viewing the structure of vesicles.For the highest quality image, select the Precise setting as the Fastest setting will be less accurate and lead to poor 3D rendering. Once the image has been generated, manipulate the 3D image by zooming and rotating to focus on a preferred location. After the view has been obtained, under the 3D tab, select Displayed Resolution, and then click on Create Image, which will create a snapshot of the image in the same orientation as it was viewed and can be saved and exported in various file formats.Confocal microscopy can be used to visualize the intracellular localization and deposition of basement membrane proteins such as Viking-GFP when the acquisition parameters are optimized. When acquisition and image processing are performed properly, super-resolution microscopy increases the image resolution compared to confocal microscopy. As illustrated by the better defined images in the intracellular trafficking of basement membrane proteins, such as Viking.Orthogonal projection allows the visualization of the overall distribution of vesicles and compartments containing basement membrane proteins in a single image using confocal or super-resolution imaging. A 3D reconstruction by assembling a stack of optical Z-sections taken by confocal or super-resolution imaging was employed to assess the localization and distribution of intracellular basement membrane proteins. Super-resolution imaging can be used to determine the precise localization of basement membrane proteins in mutant conditions.For example, in Crag knockdown epithelial cells, basement membrane proteins accumulate both apically and basally, indicating that it controls the polarized secretion of basement membrane proteins. Confocal and super-resolution microscopy can also be employed in co-localization experiments. For example, this figure shows the partial co-localization of Viking-GFP and a Golgi marker, GM-130, confirming that Viking is sorted to the Golgi before secretion.To efficiently image the trafficking of basement membrane proteins using super-resolution microscopy, we recommend to carefully mount the ovaries and take particular attention when setting the acquisition and the convolution parameters. This protocol is optimized to visualize the intracellular trafficking and secretion of basement membrane proteins. However, it can be easily modified to efficiently image the trafficking of other proteins of interest.

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