TITLE: Vidyadhara et al., 2023 “Dopamine transporter…Parkinson’s Disease&#34; (L-DOPA treatment, IHC, HPLC, Microscopy Imaging and Analysis, Western Blotting, and FSCV)
AUTHORS: Vidyadhara DJ

[DESCRIPTION]
This protocol details L-DOPA treatment, Immunohistochemistry, HPLC, and electron microscopy tissue preparation and settings and FSCV in publication  "Dopamine transporter and synaptic vesicle sorting defects underlie auxilin-associated Parkinson’s disease."

[STEPS]
SECTION: L-DOPA treatment
1. Prepare the L-DOPA treatment solution by dissolving L-DOPA powder in 0.85% saline solution to achieve a concentration of 15 mg/kg body weight. 

SECTION: L-DOPA treatment
2. Select adult mice for the study and divide them into pre-treatment and post-treatment groups.
SECTION: L-DOPA treatment
3. Conduct behavior testing on the pre-treatment group one week before the L-DOPA treatment to establish a baseline for comparison.
SECTION: L-DOPA treatment
4. On the day of the experiment, inject each mouse in the post-treatment group with a single dose of the L-DOPA treatment solution through the intraperitoneal route, at a dosage of 15 mg/kg body weight and volume of 10 ml/kg body weight.

SECTION: L-DOPA treatment
5. Conduct behavior testing on the post-treatment group 20-25 minutes after the L-DOPA treatment to measure the effect of the treatment.
SECTION: L-DOPA treatment
6. Use the pre-treatment group as a control to compare the behavioral differences before and after L-DOPA treatment.
SECTION: Immunohistochemistry
7. Anesthetize the mice using isoflurane inhalation and perfuse intracardially with 0.9% heparinized saline followed by chilled 4% paraformaldehyde (PFA) in 0.1 Molarity (M) phosphate buffer (PB).
SECTION: Immunohistochemistry
8. Post-fix the brains in the same buffer for 2880 min.

SECTION: Immunohistochemistry
9. Cryoprotect the brains in increasing grades of buffered sucrose (15 and 30%, prepared in 0.1 Molarity (M) PB), at 4 °C.

SECTION: Immunohistochemistry
10. Store the brains at -80 °C until sectioning.

SECTION: Immunohistochemistry
11. Perform serial sections of the brains (  thick) coronally using a cryostat (Leica CM1850, Germany).

SECTION: Immunohistochemistry
12. Collect the sections on gelatinized slides and store them at -20 °C.

SECTION: Immunohistochemistry: Immunoperoxidase staining
13. For immunoperoxidase staining, perform endogenous peroxidase quenching using 0.1% H2O2 in 70% methanol (30 min). 
SECTION: Immunohistochemistry: Immunoperoxidase staining
14. Block with 3% bovine serum albumin (BSA) (120 min) at Room temperature. Incubate sections at 4 °C with primary antibody (120 min at Room temperature, then 120 min).

SECTION: Immunohistochemistry: Immunoperoxidase staining
15. Incubate sections with biotin-conjugated secondary antibody at Room temperature (1:200; 3-4 hours, Vector Laboratories, PK-6101). Perform tertiary labeling with the avidin-biotin complex solution at Room temperature (1:100; 3-4 hours, Vector Laboratories, PK-6101).
SECTION: Immunohistochemistry: Immunoperoxidase staining
16. Visualize staining using 3,3′-diaminobenzidine (Fluka, 32750) as a chromogen in a solution of 0.1 Molarity (M) acetate imidazole buffer (pH 7.4) and H2O2 (0.1%).

SECTION: Immunohistochemistry: Immunofluorescence staining
17. Equilibration: Rinse the sample with 0.01 Molarity (M) PBS for 5 min, followed by rinsing with 0.01 Molarity (M) PBS + 0.1% Tx for another 5 min.

SECTION: Immunohistochemistry: Immunofluorescence staining
18. Penetration: Apply 0.01 Molarity (M) PBS + 0.5% Tx for 10-20 minutes (start with 15 minutes and adjust as needed), then wash 3 times.
SECTION: Immunohistochemistry: Immunofluorescence staining
19. Neutralization: Incubate with 0.3 Molarity (M) Glycine in 0.01 Molarity (M) PBS (without Tx) for 20 min, then wash 3 times.

SECTION: Immunohistochemistry: Immunofluorescence staining
20. Blocking: Incubate with 3% goat serum (GS) in working/wash buffer for 1.5 to 2 hours. Wash 2-3 times gently.
SECTION: Immunohistochemistry: Immunofluorescence staining
21. Primary incubation: Dilute the required concentration of primary antibody in 1% GS in working/wash buffer. Incubate for 120 min at Room temperature and 120 min at 4 °C. Wash 3-4 times.

SECTION: Immunohistochemistry: Immunofluorescence staining
22. Secondary incubation: Dilute the secondary antibody in 1% GS in working/wash buffer at a 1:500 ratio. Incubate for 2-3 hours at Room temperature. Wash 3-4 times with working/wash buffer and 2 more times with 0.01 Molarity (M) PBS (without Tx).

SECTION: Immunohistochemistry: Immunofluorescence staining
23. Mount with DAPI.
SECTION: Immunohistochemistry: Immunofluorescence staining
24. Use 1X PBS with 0.1% Triton-X-100 as both washing and working buffer for both immunoperoxidase and immunofluorescence staining, except for phospho-antibodies where 1X Tris buffer saline can be used.
SECTION: High-performance liquid chromatography (HPLC)
25. Anesthetize mice using isoflurane inhalation and sacrifice them by cervical dislocation.
SECTION: High-performance liquid chromatography (HPLC)
26. Quickly remove and dissect the brains for dorsal striatum and snap freeze them. Store the tissues at -80 °C until further use.

SECTION: High-performance liquid chromatography (HPLC)
27. Homogenize the tissues using a handheld sonic tissue dismembrator in 100-750 µL of 0.1 Molarity (M) TCA containing 0.01 Molarity (M) sodium acetate, 0.1 millimolar (mM) EDTA, and 10.5% methanol (pH 3.8).
SECTION: High-performance liquid chromatography (HPLC)
28. Spin the homogenates in a microcentrifuge at 10000 x g, 20 min and collect the supernatant for HPLC-ECD analysis.
SECTION: High-performance liquid chromatography (HPLC)
29. Estimate protein concentration using Pierce™ BCA Protein Assay Kit (Thermo Scientific).
SECTION: High-performance liquid chromatography (HPLC)
30. Use the same buffer used for tissue homogenization as the HPLC mobile phase.
SECTION: High-performance liquid chromatography (HPLC)
31. Perform HPLC using a Kinetix 2.6um C18 column (4.6 x 100 mm, Phenomenex, Torrance, CA USA).
SECTION: Unbiased stereology to count midbrain dopaminergic neurons
32. Use the optical fractionator probe of the StereoInvestigator (Software Version 8.1, Micro-brightfield Inc., Colchester, USA) connected to a brightfield microscope to perform stereological quantification of tyrosine hydroxylase (TH) positive dopaminergic neurons.
SECTION: Unbiased stereology to count midbrain dopaminergic neurons
33. Delineate the substantia nigra pars compacta (SNpc) and ventral tegmental area (VTA) on every 6th TH+ve midbrain section using a 10X objective.
SECTION: Unbiased stereology to count midbrain dopaminergic neurons
34. Identify the mounted thickness as  , which is also determined at every fifth counting site.

SECTION: Unbiased stereology to count midbrain dopaminergic neurons
35. Imply a guard zone of   on either side, thus providing   of z-dimension to the optical dissector.

SECTION: Unbiased stereology to count midbrain dopaminergic neurons
36. Count the neurons using a 40X objective with a regular grid interval of 22,500 μm² (x=150 μm, y=150 μm) and a counting frame size of 3600 μm² (x=60 μm, y=60 μm).
SECTION: Unbiased stereology to count midbrain dopaminergic neurons
37. Begin the quantification at the first anterior appearance of TH+ve neurons in the midbrain structures to the caudal most part in each hemisphere separately, which is later summed to derive total numbers.
SECTION: Electron Microscopy Tissue Preparation and Settings
38. Make sure that both freshly prepared saline and fixatives (2% PFA + 2% GA) are chilled.
SECTION: Electron Microscopy Tissue Preparation and Settings
39. Anesthetize the mouse with isoflurane inhalation in a desiccator.
SECTION: Electron Microscopy Tissue Preparation and Settings
40. Perform intracardial perfusion with chilled 0.9% heparinized saline for ~15-20 minutes (until the fluid that comes out of the heart become colorless) at a flow rate of 2 mL/minute.

SECTION: Electron Microscopy Tissue Preparation and Settings
41. Change the perfusion solution to a fixative, (2% PFA + 2% GA) (chilled) and flush it for 20-25 minutes (until tail, head and neck are stiff) at a flow rate of 2 mL/minute.

SECTION: Electron Microscopy Tissue Preparation and Settings
42. After intracardial perfusion, remove the whole brain from the skull and immerse in 2.5% glutaraldehyde + 2% PFA + 0.1 Molarity (M) cacodylate buffer, 120 min at Room temperature, followed by 120 min incubation at 4 °C (immersion fixation).

SECTION: Electron Microscopy Tissue Preparation and Settings
43. Next day, perform vibratome sectioning (  thick) of your area of interest*, using chilled 0.1 Molarity (M) PBS (in the vibratome trough).

SECTION: Electron Microscopy Tissue Preparation and Settings
44. Immediately after sectioning, transfer the   thick sections back to 2.5% glutaraldehyde + 2% PFA + 0.1 Molarity (M) cacodylate buffer and allow it equilibrate for ~30 min at 4 °C.

SECTION: Electron Microscopy Tissue Preparation and Settings
45. Wash the sections.

SECTION: Electron Microscopy Tissue Preparation and Settings
46. EM imaging can be performed using FEI Tecnai G2 Spirit BioTwin Microscope.
SECTION: Electron Microscopy Tissue Preparation and Settings
47. Images are analysed using either FIJI (NIH) or iTEM software (ResAlta Research Technologies, USA).
SECTION: Confocal/Fluorescence Microscopy and Image Analysis
48. Acquire fluorescent images using a laser scanning confocal microscope (LSM 800, Zeiss) with a 20X or 40X or 63X objectives or using a fluorescence slide scanner (VS200, Olympus) at 40X objective. 
SECTION: Confocal/Fluorescence Microscopy and Image Analysis
49. Ensure appropriate Z-depth is used.
SECTION: Confocal/Fluorescence Microscopy and Image Analysis
50. Blind all images for genotype and age before subjecting to analysis using FIJI software from National Institute of Health (NIH).
SECTION: Confocal/Fluorescence Microscopy and Image Analysis
51. After performing sum intensity projection, measure the expression intensity on an 8-bit or 16-bit image as the mean gray value on a scale of 0-255 or 0-65536, respectively.
SECTION: Confocal/Fluorescence Microscopy and Image Analysis
52. To count microglial cells, threshold images using the ‘otsu’ algorithm and count cells larger than 75-pixel units for a given image using the ‘analyze particles' function.
SECTION: Confocal/Fluorescence Microscopy and Image Analysis
53. To count immunofluorescence labeled dopaminergic neurons, use a similar method with a size threshold of 25 µm2 and above.
SECTION: Confocal/Fluorescence Microscopy and Image Analysis
54. Count astroglial cells manually using the ‘cell counter’ function.
SECTION: Confocal/Fluorescence Microscopy and Image Analysis
55. To count abnormal structures in the terminals, threshold images using the ‘triangle’ algorithm, followed by ‘analyse particles’ function. Count all the structures of size 5 µ2 and above and the circularity between 0.3 to 1.
SECTION: Confocal/Fluorescence Microscopy and Image Analysis
56. Count autophagic punctae manually using the ‘cell counter’ function, which can be further validated on thresholded images using ‘triangle’ algorithm, followed by ‘analyse particles’ function (size: 0.5 µ2, circularity: 0.0 to 1).
SECTION: Confocal/Fluorescence Microscopy and Image Analysis
57. Perform colocalization analysis using the “Coloc 2” function.
SECTION: Western blotting
58. Homogenize the samples using an appropriate buffer and determine their protein concentration using a protein assay (such as the Bradford or BCA assay). Adjust the protein concentration of the samples to the desired amount using the buffer. 
SECTION: Western blotting
59. Heat the samples at 95 °C for 10 min if fresh, or 5 minutes if previously heated, to denature the proteins.

SECTION: Western blotting
60. Load precast or poured polyacrylamide gels with the desired concentration and number of wells. Ensure that the gel is completely polymerized before loading the samples.
SECTION: Western blotting
61. Run the gels at 65 V through the stacking gel for about 30-45 minutes until the dye front reaches the top of the stacking gel. Then, increase the voltage to 120 V and run until the dye front reaches the bottom of the resolving gel or runs off the gel.
SECTION: Western blotting
62. Incubate the PVDF membrane in 100% methanol for 1 min on a shaker to activate the membrane. Discard the methanol and rinse the membrane with distilled water.

SECTION: Western blotting
63. Incubate the activated membrane in distilled water for 1 min on a shaker to rehydrate the membrane.

SECTION: Western blotting
64. Soak the membrane in transfer buffer for 10-15 minutes prior to use to equilibrate the membrane with the buffer.
SECTION: Western blotting
65. Assemble the transfer sandwich by placing the gel on top of the anode (black side), followed by a sponge soaked in transfer buffer, a piece of transfer paper, the PVDF membrane, another piece of transfer paper, a sponge soaked in transfer buffer, and finally the cathode (white side). 
 

SECTION: Western blotting
66. Transfer the proteins from the gel to the membrane by running the transfer at 4 °C at constant 400 mAmps for 240 min (60 min) or at constant 100 V for 60 min, depending on the properties of the protein of interest.

SECTION: Western blotting
67. After transfer, incubate the blots in 6 mL of blocking solution (such as 1X TBS + 5% non-fat milk + 5% goat serum) for 60 min at Room temperature on a rocker to block nonspecific binding sites.

SECTION: Western blotting
68. Incubate the blots 60 min on a rocker at 4 °C in the primary antibody diluted in 1X TBS + 5% non-fat milk + 5% goat serum. 
 

SECTION: Western blotting
69. Wash the blots with 1X TBST (TBS + 0.1% Tween-20) to remove unbound primary antibody.
SECTION: Western blotting
70. Incubate the blots for 60 min at Room temperature on a rocker in the secondary antibody diluted in 1X TBST + 5% non-fat milk + 5% goat serum (optional: add 5 µL of 10% SDS to the secondary antibody solution).

SECTION: Western blotting
71. Wash the blots 3 times for 5 minutes each with 1X TBST to remove unbound secondary antibody. 
72. Image the blots using an appropriate detection method (such as chemiluminescence or fluorescence) and a suitable imaging system. Store the blots in TBS buffer or dry them and store them between filter papers in a light-protected bag.
SECTION: Surgery and in vivo Fast Scanning Cyclic Voltammetry (FSCV)
73. Anesthetize mice using isoflurane (SomnoSuite Small Animal Anesthesia System, Kent Scientific; induction 2.5%, maintenance 0.8–1.4% in O2, 0.35 l/min). 
SECTION: Surgery and in vivo Fast Scanning Cyclic Voltammetry (FSCV)
74. Fix the head of the mouse on a stereotaxic frame (Kopf Instruments, Tujunga, CA).
SECTION: Surgery and in vivo Fast Scanning Cyclic Voltammetry (FSCV)
75. Apply Puralube vet ointment on the eye to prevent cornea from drying out.
SECTION: Surgery and in vivo Fast Scanning Cyclic Voltammetry (FSCV)
76. Use a stereotactic drill (0.8 mm) to perform a craniotomy (unilateral, right) to target the midbrain and dorsal striatum with the following coordinates (values are in mm from Bregma):
SECTION: Surgery and in vivo Fast Scanning Cyclic Voltammetry (FSCV)
77. Place an Ag/AgCl reference electrode via a saline bridge under the skin.
SECTION: Surgery and in vivo Fast Scanning Cyclic Voltammetry (FSCV)
78. Lower a 22G bipolar stimulating electrode (P1 Technologies, VA, USA) to target ventral midbrain (between 4-4.5mm). Adjust the exact depth for maximal dopamine release.
SECTION: Surgery and in vivo Fast Scanning Cyclic Voltammetry (FSCV)
79. Lower a custom-built carbon fiber electrode (5 μm diameter, cut to ∼150 μm length, Hexcel Corporation, CT, USA) to reach dorsal striatum for recording the evoked dopamine release.
SECTION: Surgery and in vivo Fast Scanning Cyclic Voltammetry (FSCV)
80. Use dil-coated carbon-fiber electrodes to identify the electrode position in the dorsal striatum and the electrode track in the brain tissue to identify the position of the stimulation electrode.
SECTION: Surgery and in vivo Fast Scanning Cyclic Voltammetry (FSCV)
81. Measure the evoked dopamine release using constant current (400μA), delivered using an Iso-Flex stimulus isolator triggered by a Master-9 pulse generator (AMPI, Jerusalem, Israel). A single burst stimulation consists of 30 pulses at 50Hz (0.6s).
SECTION: Surgery and in vivo Fast Scanning Cyclic Voltammetry (FSCV)
82. Calibrate electrodes using a known concentration of dopamine in artificial cerebrospinal fluid (ACSF).
SECTION: Surgery and in vivo Fast Scanning Cyclic Voltammetry (FSCV)
83. Use IGOR Pro for the data acquisition and analysis. 
 

SECTION: Electron Microscopy Tissue Preparation and Settings
45.1. Wash the sections in 0.1 Molarity (M) cacodylate buffer for 2 min. (1/3)

SECTION: Electron Microscopy Tissue Preparation and Settings
45.2. Wash the sections in 0.1 Molarity (M) cacodylate buffer for 2 min. (2/3)
SECTION: Electron Microscopy Tissue Preparation and Settings
45.3. Wash the sections in 0.1 Molarity (M) cacodylate buffer for 2 min. (3/3)
SECTION: Electron Microscopy Tissue Preparation and Settings
45.4. Then, place the sections in the same buffer and deliver them to EM core for further processing.
SECTION: Western blotting
69.1. Wash the blots for 5 min with 1X TBST. (1/3)

SECTION: Western blotting
69.2. Wash the blots for 5 min with 1X TBST. (2/3)
SECTION: Western blotting
69.3. Wash the blots for 5 min with 1X TBST. (3/3)
71.1. Wash the blots for 5 min each with 1X TBST. (1/3)

71.2. Wash the blots for 5 min each with 1X TBST. (2/3)
71.3. Wash the blots for 5 min each with 1X TBST. (3/3)
71.4. Rinse the blots with TBS to remove residual Tween-20.
SECTION: Surgery and in vivo Fast Scanning Cyclic Voltammetry (FSCV)
76.1. Midbrain: anteroposterior = -2.9, mediolateral=+1.0, dorsoventral=+4
SECTION: Surgery and in vivo Fast Scanning Cyclic Voltammetry (FSCV)
76.2. Dorsal Striatum: anteroposterior = +1.2, mediolateral = +1.3, dorsoventral = +3.1