Vectasheild mounting media

The central nervous system was dissected out of 30-day-old adult Drosophila, across indicated genotypes. All fat bodies and trachea were removed from the nervous tissue because it is auto-fluorescent. Tissue was adhered to a glass coverslip with Vectabond (Vector labs) and fixed in 1% PFA overnight at 4 °C. 1% PFA in PBS was prepared fresh, as premixed PFA in a methanol stabilizer disrupts ApopTag assays. Post-fix processing was performed in Ethanol:Acetic acid (2:1) for 10 min at −20 °C. ApopTag assay was performed as described in Millipore Sigma ApopTag Fluorescein kit protocol. Tissue was mounted in vectasheild mounting media (Vector Labs) containing DAPI as a counterstain to image nuclei in the sample. ApopTag Fluorescein signal was imaged in the FITC channel. Samples were imaged on a Nikon Ti-E spinning disc confocal microscope with an Andor Zyla 5.2 camera, using micromanager software. Images were captured with a Plan Apo λ ×20/0.75 Nikon objective. ApopTag Fluorescein positive nuclei were counted using FIJI 3D Object counter.

Tissues dissected from third instar larvae were fixed and stained as Ciurciu et al.,^{54 (link)} with minor modifications. After fixation for 20 min in 4% (w/v) paraformaldehyde in PBS, dissected brains from third instar larvae were washed in PBS with 0.1% Triton-X (PBST), then blocked for 2 h in PBST with 5% FCS (blocking solution). Primary antibody staining was done overnight at 4°C in blocking solution, washed three times with PBST and incubated with secondary antibody in blocking solution for 2 h at room temperature. After three washes in PBST, brains were mounted in Vectasheild mounting media (Vector laboratories, Peterborough, UK). Primary antibodies were as follows: rabbit anti-Laminin (1:1000); guinea-pig anti-Repo (1:1000); rabbit anti-Repo (1:25,000); mouse anti-NimC1 P1^{55 (link)}(1:30), mouse anti-Mmp1 (1:1:1 mix of 3A6B4, 3B8D12, 5H7B11 from Developmental Studies Hybridoma Bank, Iowa, USA diluted 1:10); mouse anti-β−gal (Promega, Southampton, UK, 1:100); mouse anti-SRF (Active Motif, La Hulpe, Belgium, 1:100). Secondary antibodies were conjugated to Alexa-Fluor 555 or 633 (Invitrogen, Paisley, UK, 1:500). TO-PRO-3 Iodide (Invitrogen, 1:1000) or DAPI was used to visualize DNA.