Regulation of mitophagy by the NSL complex underlies genetic risk for Parkinson’s disease: Drosophila Husbandry, Assays and Immunohistochemistry
Natalie J. Welsh, Alexander J. Whitworth
Abstract
This protocol describes Drosophila stocks and husbandry, Locomotor and lifespan assays and, Immunohistochemistry and sample preparation in order to assess the role of KAT8/KANSL1 in neuronal survival in vivo .
Steps
Drosophila stocks and husbandry
Raise flies under standard conditions in a humidified, temperature-controlled incubator with a 12h:12h light:dark cycle at 25°C, on food consisting of agar, cornmeal, molasses, propionic acid and yeast.
Locomotor Assay
Perform the startle induced negative geotaxis (climbing) assay using a counter-current apparatus.
Place 20-23 males into the first chamber, and tap to the bottom.
Give 10 s to climb a 10 cm distance.
Repeat this procedure five times (five chambers), and count the number of flies that remain in each chamber.
Normalize the weighted performance of several group of flies for each genotype to the maximum possible score and expressed as Climbing index as described previously in the manuscript by Greene et al., 2003.
Lifespan Assay
For lifespan experiments, grow flies at low-density under identical conditions as described in Step 1 .
Collect progeny under very light anaesthesia and keep in tubes of approximately 20 males each, around 50-100 in total.
Transfer flies every 2-3 days to fresh medium and record the number of dead flies.
Calculate percent survival at the end of the experiment after correcting for any accidental loss.
Sample Preparation and Immunohistochemistry
Dissect drosophila brains from aged flies and immunostain as previously described in the manuscript by Whitworth et al., 2005.
Dissect adult brains in PBS and fix in 4% (w/v) formaldehyde for0h 30m 0s On ice.
Permeabilize in 0.3% (v/v) Triton X-100 for 3 times 0h 20m 0s.
Block with 0.3% (v/v) Triton X-100 plus 4% (v/v) goat serum in PBS for 4h 0m 0s at Room temperature.
Incubate tissues with anti-tyrosine hydroxylase (Immunostar Inc. #22491), diluted in 0.3% (v/v) Triton X-100 plus 4% (v/v) goat serum in PBS for 72h 0m 0s at 4°C, then rinse 3 times 0h 20m 0s with 0.3% (v/v) Triton X-100 in PBS, and incubate with the appropriate fluorescent secondary antibodies 0h 20m 0s at 4°C.
Wash tissues 2 times in PBS and mount on slides using Prolong Diamond Antifade mounting medium (Thermo Fisher Scientific).
Image brains with a Zeiss LMS 880 confocal. Count tyrosine hydroxylase-positive neurons under blinded conditions.
