A Simple Immunofluorescence Method to Characterize Neurodegeneration and Tyrosine Hydroxylase Reduction in Whole Brain of a Drosophila Model of Parkinson’s Disease

Materials and reagents

1. Drosophila melanogaster [Oregon K (OK) flies, procured from the National Drosophila Stock Center, Mysore University, Mysuru, India, were raised in food media containing sucrose, yeast, agar-agar, and propionic acid, and maintained in a fly environment chamber and used in the current study [3,10,13,28]]. Depending on the nature of the experiment, flies with and/or without neurotoxicant treatment (sporadic PD model) and transgenic fly model(s) can be employed.

2. Sterilized 1.5 mL centrifuge tubes (Tarsons, catalog number: 500010)

3. Parafilm^TM wrapping film (Bemis, catalog number: PM996)

4. Conical flask (Borosil, catalog number: 5100)

5. Magnetic stirrer bar #8 mm × 40 mm (Tarsons, catalog number: 4113)

6. SPINNOT^TM digital magnetic stirrer hotplate (Tarsons, catalog number: 6090)

7. Sterilized micro tips (Tarsons, catalog number: 521010)

8. Freshwrapp aluminum foil 9–11 μm (Hindalco, catalog number: HV2241)

9. Glass plate (Suwimut, catalog number: B08FRB2NTM)

10. Fingernail polish (FacesCanada, catalog number: CC4403)

11. Glass spacer (Borosil, catalog number: 9115S01)

12. Microscopy slides #76 mm × 26 mm (ReliGlas, catalog number: 7101)

13. Gold-seal coverslips (22 mm²) (Electron Microscopy Sciences, catalog number: 63765-01)

14. Sucrose (SRL, catalog number: 84973)

15. Agar-agar (HiMedia, catalog number: GRM666)

16. Sugar-tolerant yeast (Angel, catalog number: 5331A201908)

17. Propionic acid (Merck, catalog number: 80060505001730)

18. Whatman^TM filter paper (GE Healthcare, catalog number: 1001917)

19. Paraformaldehyde (PFA) pH 7.4 (Sigma-Aldrich, catalog number: I58127)

20. Phosphate buffered saline (PBS) pH 7.4 (HiMedia, catalog number: ML023)

21. Triton X-100 (Sigma-Aldrich, catalog number: T8787)

22. Normal goat serum (NGS) (Vector Labs, catalog number: S1000)

23. Rabbit anti-tyrosine hydroxylase (anti-TH) polyclonal primary ab (Millipore, catalog number: Ab152)

24. Goat anti-rabbit IgG H&L (TRITC-labeled) polyclonal secondary ab (Abcam, catalog number: Ab6718)

25. VECTASHIELD^® mounting medium (Vector Labs, catalog number: H1000)

1. 4% PFA solution (50 mL) (see Recipes)

2. 0.1% PBST (phosphate buffered saline and Triton X-100) (50 mL) (see Recipes)

3. 0.5% PBST (50 mL) (see Recipes)

4. 5% NGS blocking buffer solution (1 mL) (see Recipes)

5. Anti-TH polyclonal primary ab solution (see Recipes)

6. TRITC-labeled polyclonal secondary ab solution (see Recipes)

Recipes

1. 4% PFA solution (50 mL)

   PFA 2 g

   1× PBS 50 mL

   1. Add PFA in 1× PBS in a conical flask, cover it with parafilm, and shake it thoroughly for 10 min.

   2. Transfer the flask with a magnetic stirrer on the hotplate for heating/boiling with a temperature ranging from 80 °C to 110 °C with moderate stirring at 150 rpm.

   3. Keep the flask on the hotplate until the cloudy solution becomes transparent.

   4. After this, switch off the hotplate but keep the stirring for 15 min. Allow the solution to cool down, aliquot it in a 1.5 mL centrifuge tube, and store it at -80 °C.

   Critical: Do not store the solution for more than a week.

   Caution: PFA is a potential carcinogen; hence, the whole process should be done under a fume hood. Wear hand gloves and a lab coat during handling and preparation of PFA solution.




2. 0.1% PBST (phosphate buffered saline and Triton X-100) (50 mL)

   10× PBS 5 mL

   Autoclaved enzyme-free water 45 mL

   Triton X-100 50 μL

   1. Add 5 mL of 10× PBS in 45 mL of autoclaved enzyme-free water.

   2. Mix 50 μL of Triton X-100 and vortex it for 10 seconds. The solution can be stored at room temperature for one week.




3. 0.5% PBST (50 mL)

   10× PBS 5 mL

   Autoclaved enzyme-free water 45 mL

   Triton X-100 250 μL

   1. Add 5 mL of 10× PBS in 45 mL of autoclaved enzyme-free water.

   2. Mix 250 μL of Triton X-100 and vortex it for 10 s. The solution can be stored at room temperature for one week.




4. 5% NGS blocking buffer solution (1 mL)

   NGS 50 μL

   0.5% PBST 950 μL

   Add 50 μL of NGS in 950 μL of 0.5% PBST and mix it properly by vortexing for 10 s. The solution can be stored at room temperature for 1–2 h.




5. Anti-TH polyclonal primary ab solution

   Anti-TH polyclonal primary ab 5 μL

   5% NGS blocking buffer 1,245 μL

   Take 1,245 μL of 5% NGS blocking buffer and add 5 μL of anti-TH polyclonal primary ab (1:250 dilution). Mix it gently by inverting the tube slowly and place it on the ice until used.




6. TRITC-labeled polyclonal secondary ab solution

   TRITC-labeled polyclonal secondary ab 5 μL

   5% NGS blocking buffer 1,245 μL

   Take 1,245 μL of 5% NGS and add 5 μL of TRITC-labeled polyclonal secondary ab (1:250 dilution). Mix it gently by inverting the tube slowly and store it on ice until used.

Equipment

1. Fly head capsule handling items e.g., needles #31 G × 6 mm (Tentabe BD, catalog number: 324902)

2. Dissecting fine forceps (EMS, catalog number: 78620-4B)

3. Brush (TEYUP, model number: SR-1013)

4. Delicate task Kim wipers (KIMTECH^TM, catalog number: 370080)

5. Micropipette i.e., 1,000 μL, 50 μL, 10 μL, 2 μL (Gilson, catalog number: 30040)

6. Frost-free refrigerator (Whirlpool, model number: FF26 4S)

7. pH/mV meter (Hanna Instruments, model: HI2211-02)

8. -20 °C ES Series refrigerator (Thermo Scientific, model: 50616100444443250)

9. -80 °C ultra-low temperature freezer (New Brunswick Innova, model: U101-86)

10. Stereo zoom microscope (Carl Zeiss, model: Stemi 305)

11. Stereo zoom microscope (Leica, model: E24)

12. Fume hood (BIOMATRIX, Telangana, India)

13. BOD incubator (Percival, model: DR-36VL)

14. Test tube rotator (Tarsons, Rotospin, catalog number: 3070) and disk for 24 × 1.5 mL tube (Tarsons, catalog number: 3071)

15. Axio Imager M2 fluorescence microscope fitted with 100 W Mercury lamp (Carl Zeiss, catalog number: 430004-9902-000)

16. AxioCam ICm1 monochromatic camera (Carl Zeiss, catalog number: 426553-9901-000)

Software and datasets

1. ZEN 2012 SP2 blue edition, version 2.0.14283.302 (Carl Zeiss, Jena, Germany)

2. Microsoft Office Excel Worksheet 2007 (Microsoft Inc., WA, USA)

3. GraphPad Prism, March 7, 2007, version 5.00 (GraphPad Inc., MA, USA)

Procedure