In vivo Assessment of Lysosomal Stress in the Drosophila Brain Using Confocal Fluorescence Microscopy

Materials and Reagents

1. 1.7 mL microtubes (Axygen, catalog number: MCT-175-C)

2. 48-well Nunc cell plate (Thermo Scientific, catalog number: 150687)

3. Micropipette 200 µL tips (Axygen, catalog number: T-200-Y)

4. Double-sided tape (Scotch 665 12.7 mm × 22.8 m)

5. Aluminum foil

6. Microscopy slides (Westlab, catalog number: 663-249)

7. Cover slips (Trajan, catalog number: 471112440M)

8. Fly stocks of interest

9. LysoTracker^TM Red DND-99 (Thermo Fisher Scientific, catalog number: L7528)

10. LysoTracker^TM Blue DND-22 (Thermo Fisher Scientific, catalog number: L7525).

11. LysoTracker^TM Green DND-26 (Thermo Fisher Scientific, catalog number: L7526).

12. Phosphate-buffered saline (PBS) (Sigma-Aldrich, catalog number: P5493)

13. Schneider's Drosophila medium 1× (Thermo Fisher Scientific, catalog number: 21720024)

14. Spinosad (Sigma-Aldrich, catalog number: 33706)

15. Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: 276855-100ML)

16. Vectashield 10 mL mounting medium (Vector, catalog number: VEH1000)

17. Analytical standard sucrose (Merk, product number: 47289)

18. 5% sucrose solution (200 mL) (see Recipes)

19. 1,000 ppm spinosad solution in DMSO (see Recipes)

20. 5× spinosad stock solution (see Recipes)

21. 5× DMSO stock solution (see Recipes)

22. LysoTracker 1:100 working stock solution (see Recipes)

23. Distilled water

Equipment

1. Dissecting forceps (Fine Science Tools, Dumont #5 Forceps, catalog number:1195-10)

2. Stereo microscope (Zeiss, model: Stemi 2000-C)

3. Confocal fluorescence microscope (Leica TCS SP8, DM600 CS)

4. Micropipette P200

5. 500 mL laboratory bottles

6. Nutating mixer (Corning, catalog number: 6720)

Software

1. Fluorescence microscope image acquisition software (LAS X)

2. ImageJ/FIJI (NIH, https://fiji.sc/)

3. GraphPad Prism (Dotmatics, http://www.graphpad.com)

Procedure

1. Larvae collection and insecticide exposure

   1. Using the micropipette, load a 48-well Nunc plate with 200 µL of 5% sucrose solution per well.

   2. Using a pair of forceps, gently transfer 25 early third-instar larvae per well (Figure 1A, B).

   3. For the insecticide exposure, add 50 µL of the 5× spinosad stock solution (see Recipes) to each well containing larvae to be exposed and give the plate a gentle swirl. For unexposed controls, add 50 µL of the 5× DMSO stock solution instead (see Recipes). The final concentration of insecticide used here is 2.5 ppm.

   4. Keep the plate at 25°C and covered with aluminum foil to protect it from light for 2 h (or desirable number of hours), until exposure is over.

   5. Once exposure time is over, using a pair of forceps gently transfer larvae to a new well containing 250 µL of 1× PBS.

      
      

2. Tissue dissection and staining

   1. Transfer larvae from the PBS-containing well to a microscopy slide containing a drop of cold (4°C) Schneider's medium 1×.

   2. Under the stereo microscope, begin the dissection. Using two pairs of forceps, hold the larval body at the midpoint (Figure 1C) and pull the anterior and posterior regions apart (Figure 1D).

   3. Using a pair of forceps, gently transfer the anterior body section (Figure 1E) to a microtube containing 495 µL of cold (4°C) 1× PBS.

   4. Add 5 µL of LysoTracker Red working stock solution (see Recipes) to the microtube. Final LysoTracker concentration of 1:10,000 (Figure 1F).

   5. Wrap the microtube in aluminum foil to protect it from light and keep it at slow agitation on a nutator mixer for 7 min (Figure 1G).

   6. Using a micropipette, slowly and completely remove the staining solution without touching the sample (Figure 1H).

   7. Gently add 500 µL of 1× cold (4°C) PBS back into the microtube (Figure H) and place it back on the nutator mixer for 5 min (Figure I).

   8. Under the stereo microscope, add a drop of cold (4°C) Schneider's medium 1× to a new microscopy slide. Using a pair of forceps, gently transfer the stained sample from the microtube onto the slide (Figure 1J).

   9. Conclude the dissection by holding the anterior body section sideways with two pairs of forceps and gently pull them apart from each other to tear the cuticle. Once the brain is located, use a pair of forceps to clear the surrounding tissues (Figure 1K).

      
      

3. Mounting slides for fluorescence microscopy

   1. Add a small drop of Vectashield on top of the brain, only enough to cover it. Using a pair of forceps, if necessary, adjust sample orientation.

   2. Cut two small pieces of double-sided tape and place them flanking the sample in Vectashield.

   3. Place the coverslip on the top, aligning its border with the double-sided tape (Figure 1L).

      
      

   
   Figure 1. Experimental procedure. A. 48-well Nunc plate with 200 µL of 5% sucrose solution and 25 larvae per well. B. Detail of well containing 25 early third-instar larvae. C. For partial dissection start by holding down a larva at midpoint. D. Pull anterior and posterior body regions apart. E. Larva anterior body region. F. Transfer the anterior body region to a microtube containing cold 1× PBS and add LysoTracker Red working stock solution to it. G. Wrap the microtube in aluminum foil and keep it at slow agitation on a nutator mixer for 7 min. H. Perform one washing step by replacing the solution in the microtube with cold 1× PBS. I. Return the microtube wrapped in foil to the nutator mixer at low agitation for 5 min. J. Transfer the partially dissected sample to a slide containing a drop of cold Schneider’s medium. K. Under the microscope, conclude dissection, and isolate the brain from other tissues. L. Mount the slide for microscopy using double-sided tape, Vectashield, and a cover slip and proceed to image acquisition immediately.

   
   

4. Imaging

   1. Proceed to image acquisition with a confocal fluorescence microscope immediately. Given that no fixative solution is used here, time from partial dissection to acquisition of last image should be kept within 30 min to avoid tissue degradation.

   2. In some cases, LysoTracker staining may not penetrate the more central parts of the brain or may aggregate at the brain’s surface. For that reason, to maintain consistency across samples, it is desirable to acquire images from a zone that excludes the brain’s surface and avoids central areas (Figure 2A).

   3. To ensure consistency and accuracy in the measurements, set optimal values of laser power and gain with a control sample and maintain the same settings across all samples.

   4. Select the same zones for imaging across all samples. Here, to maintain consistency, images were only acquired from the optic lobes.

   5. Once the zone is localized, acquire red signal (excitation/emission 577/590 nm) using a 40× objective (NA 1.1). It is important to find consistency in the signal observed within control samples, as well as exposed samples. Lack of consistency may indicate sample degradation or inappropriate staining.

   6. Acquire at least 20 images across the z-stack for every sample with 1 µm distance between them and avoid imaging the sample’s top and bottom. Image at least seven brain samples per group/treatment condition.

Data analysis

1. Analyzing microscopy images on ImageJ

   1. Open the image files on ImageJ and, using the rectangle selection tool, create a region of interest (ROI) of 30 × 30 µm (Figure 2A). The ROI will be used to quantify the percentage of area occupied by LysoTracker.

   2. Right click on the rectangle and duplicate the selected area.

   3. On top bar menu, click Image > Adjust > Threshold. A new window will appear; select Dark background. If necessary, adjust the threshold bar to reduce background. Take note of the percentage of area occupied by LysoTracker (Figure 2B).

   4. Acquire three to five independent ROIs, randomly assigned across the z-stack and within the zone, to each sample. Measure the percentage of area occupied by LysoTracker for each ROI in the same way.

   5. LysoTracker is also available in the options blue and green.

      
      

2. Statistical analysis and plotting

   1. Make an average of the independent measurements acquired per sample. Use these averaged values for plotting graphs and performing statistical analysis.

   2. Using a statistics software of your preference (such as GraphPad Prism), perform a Student’s unpaired t-test. Plot results as a bar plot including the dot plot for individual values (Figure 2C).

      
      

   
   Figure 2. Image analysis and data representation. A. Acquire images of the brain zone (i.e., excluding the brain’s surface and avoiding central areas) from where regions of interest (ROIs) will be later selected. B. Duplicate a 30 × 30 µm ROI and, using the threshold tool, measure the percentage of area occupied by LysoTracker (small red box). 400× magnification. C. LysoTracker area (%) (n = 7 brain samples/treatment; three image sections/brain), Student’s unpaired t-test, p-value < 0.0001.

Notes

1. Given the exposure method used here, early third-instar larvae are preferable, as late wandering third instars may crawl out of the wells or start pupation during insecticide exposure.

2. Start the exposure of each well at regular intervals to accommodate time to perform dissections and microscopy. This will optimize the number of samples that can be assessed per experimental batch.

Recipes

1. 5% sucrose solution (200 mL)

   Distilled water 190 mL

   Analytical standard sucrose 10 g

   Into a 500 mL glass bottle, add 190 mL of water and 10 g of sucrose. Give the bottle a swirl until completely dissolved. The solution can be stored at 4°C for up to one month. Solution must be at room temperature for usage.

   
   

2. 1,000 ppm spinosad solution in DMSO

   DMSO 1 mL

   Spinosad 1 mg

   Add 1 mL of DMSO and 1 mg of spinosad into a 1.7 mL microtube and vortex it vigorously for a few minutes until solubilized. Create ten 100 µL aliquots in separated microtubes and store them at -20°C. Avoiding multiple freeze-thaw cycles and protecting the solutions from light will increase the insecticide’s half-life. Let the solution thaw at room temperature before usage.

   
   

3. 5× spinosad stock solution

   1,000 ppm spinosad solution in DMSO 12.5 µL

   5% sucrose solution 987.5 µL

   Add 987.5 µL of 5% sucrose solution and 12.5 µL of the 1,000 ppm spinosad solution in DMSO into a 1.7 mL microtube and vortex for 30 s. This will create a 12.5 ppm spinosad solution that will be used to dose the wells of the Nunc plate where larvae are to be exposed.

   
   

4. 5× DMSO stock solution

   DMSO 12.5 µL

   5% sucrose solution 987.5 µL

   Add 987.5 µL of 5% sucrose solution and 12.5 µL of DMSO into a 1.7 mL microtube and vortex it for 30 s. This will create a 12.5 ppm DMSO solution that will be used to generate the control exposure.

   
   

5. LysoTracker 1:100 working stock solution

   1× PBS 495 µL

   LysoTracker^TM Red DND-99 5 µL

   Thaw one LysoTracker^TM Red DND-99 vial at room temperature. To a 1.7 mL microtube add 495 µL of 1× PBS and 5 µL of LysoTracker^TM Red DND-99. Using a micropipette, gently mix the solution. Wrap the microtube in foil to protect it from light and keep it at room temperature. Prepare it immediately before usage.

Acknowledgments

The author was supported by a Victorian Latin America Doctoral Scholarship (Victorian Government and the University of Melbourne), an Alfred Nicholas Fellowship (University of Melbourne), and a University of Melbourne Faculty of Science Travelling Scholarship.

Original research paper:

Martelli, F., Hernandes, N. H., Zuo, Z., Wang, J., Wong, C. O., Karagas, N. E., Roessner, U., Rupasinghe, T., Robin, C., Venkatachalam, K., et al. (2022). Low doses of the organic insecticide spinosad trigger lysosomal defects, elevated ROS, lipid dysregulation, and neurodegeneration in flies.Elife 11: e73812.

Competing interests

The author declares no conflicts of interest.

Ethics

Genetically modified Drosophila strains were maintained in a physical containment insectary facility following the biosafety guidelines by the Office of Research Ethics & Integrity at the University of Melbourne.

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