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.
