Read me File for ‘Developing a Drosophila–based model in which to study aspects of pathological Tau transfer and seeding’ Dataset DOI: 10.5258/SOTON/D2819? Date that the file was created October, 2023 ------------------- GENERAL INFORMATION ------------------- ReadMe Author: Ben Batchelor, University of Southampton, ORCID ID 0009-0009-6722-9874 Date of data collection: 2018-2022 Data collected in Building 85, University of, Southampton SO17 1BJ Grant funding: Gerald-Kerkut Trust code 500085151 -------------------------- SHARING/ACCESS INFORMATION -------------------------- Licenses/restrictions placed on the data, or limitations of reuse: Creative Commons Attribution CC-BY This dataset supports the thesis publication: Authors: Ben Batchelor Title: ‘Developing a Drosophila–based model in which to study aspects of pathological Tau transfer and seeding’ -------------------- DATA & FILE OVERVIEW -------------------- This dataset contains: [Chapter_4.zip (Figure 13, Figure 14, Figure 15, Figure 16, Figure 17), Chapter_5.zip (Figure 22, Figure 23, Figure 24, Figure 25, Figure 26, Figure 27, Figure 28) Chapter_6.zip (Figure 33, Figure 34, Figure 35, Figure 36) and Appendix.zip] Data saved here consists of the Images and Graphpad prism files (raw data, analysis and graphs) used in the creation of the thesis: Developing a Drosophila–based model in which to study aspects of pathological Tau transfer and seeding. Organised by results chapter and figure all images and graphs from the thesis are here. Image files are Tifs and show Drosophila brains imaged with Confocal microscopy. These brains contain fluorescently tagged Tau and GFP. Using Immunohistochemistry these proteins are targeted by DAKO anti-Tau antibodies or anti-GFP antibodies respectively with a corresponding fluorescent secondary antibody. Other antibodys include markers of toxic Tau species (AT8 targeting hyperphosphorylation and MC1 targeting the misfolded phenotype) and Drosophila synaptic active zone protein nc82 Graphpad prism files contain the raw input values (e.g. area measured in imageJ). These files also contain the statistical analysis and parameters used alongside the final graphs displayed. -------------------------- METHODOLOGICAL INFORMATION -------------------------- Assays were conducted by crossing the appropriate UAS and Gal4 lines and collecting male progeny bearing the correct markers. Collection of male offspring was carried out whilst the flies were still virgins and for zero day time points flies were dissected immediately in order for the T=0 timepoint to be as close to eclosion as possible. All Drosophila were raised on standard Bloomington media at 23°C in a 12/12 hour light/dark cycle unless otherwise stated hereafter For Longevity Assays Male offspring which had eclosed within 0-3 days were isolated from each genotype, forming ten groups of ten flies per individual genotype. The number of dead flies in each vial was assessed three times a week and kept on standard Bloomington media at 23°C and 12/12 hour light/dark cycle. The surviving flies were moved to a new vial of food once a week. The recorded number of surviving flies was used to carry out a survival analysis using GraphPad Prism where a Kaplein-Meir curve was plotted to determine median life expectancy. A Mantel-cox Log-rank test was then used to determine the significance of any differences in survivability. For Injections Progeny from the experimental stocks were selected for both sex and correct marker gene expression. Males were isolated into groups of ten and aged until appropriate time points (0, 3, 7 and 14 days) at which point they were anesthetised with CO2. Once anesthetised, a small nick was made in the right-hand side 2nd or 3rd antennal lobe using dissecting scissors (see Figure 9 below). A pulled capillary tube needle was then placed into this nick. Capillary action drew the contents of the needle into the 3rd antennal lobe. The fly was then transferred immediately to a fresh food vial to recover and age to the appropriate time point. For Dissections Progeny from the experimental stocks were selected for both sex and correct marker gene expression. Males were isolated into groups of ten and aged until appropriate time points (0, 3, 7 and 14 days), at which point they were anesthetised with CO2. Individuals were decapitated using a pair of tweezers (Fisherbrand™ Dumont #5 Fine Tip Tweezers) to pull on the flies’ proboscis. The heads were then immediately transferred to a watch glass with 100?l of PBST and dissected at room temperature by gripping the carapace in the gap left by the removal of the proboscis and peeling back tissue surrounding the brain. The trachea and remaining eye pigment were removed before the dissected brain was placed in 4% PFA for twenty minutes with gentle agitation. After 20 minutes, the PFA was removed and the brains washed three times for 20 minutes with 1000?l of PBST. For Immunohistochemistry (IHC) All steps were carried out at room temperature with gentle agitation. Dissected brains were blocked in 3% NGS for one hour and then a primary antibody (Table 3) was added and left overnight at room temperature. The brains were then washed three times for 10 minutes in 1000?l phospho-buffered saline with Triton X-100 (PBST) before being re-suspended in 200?l of 3% normal goat serum (NGS) and the appropriate secondary antibody (Table 3) for 2 hours. After this, the secondary antibody was washed off with a brief PBS wash. For Confocal imaging Confocal microscopy was chosen due to the highly 3D structure of the fly brain, requiring a technique that allows for optically sectioning of tissue. A pinhole was used to block out of focus light, permitting an optical slice of the specimen to be taken. Multiple optical slices taken by confocal microscopy can be built up into z-stacks, allowing for a 3D reconstruction of the fly brain in ImageJ. The use of multiple detectors and sequential scanning also allows for the separation of multiple fluorescently- tagged proteins and stains within the sample. Using a Leica SP8 Inverted scanning confocal microscope, 12-bit depth stacks were taken across the whole brain at either 20x (dry) or 63x (oil) magnification. These stacks were taken at 1024x1024 resolution at 400Hz line average 2. Sequential excitation steps were used to minimize bleed through between channels which were detected on a Leica HyD hybrid detector. Image processing was carried out using ImageJ Fiji 1.53e to apply colour tables and scale bars. For Analysis The area coverage of GFP, mCherry and MC1 was measured using (Fiji is Just) ImageJ 1.53e by thresholding the channel using auto-setting and adjusting to remove excess background. This allowed for a measurement of the area coverage of signal. Measurements of fluorescence were avoided due to conditions during slide preparations causing a variability in this signal, given that some of these experiments occurred across multiple weeks. Area coverage is also more relevant to Tau spread as it should only increase as Tau moves into new neuronal populations. However, some of this signal will be attributable to Tau signal moving throughout (e.g. from somatodendritic to axonal). For Statistics Differences between the area coverage of tested fluorophores was analysed using a t-tests and one-way ANOVAs followed by Dunnett’s multiple comparisons test or two-way ANOVAs followed by Tukeys mixed-effects analysis. These were performed using GraphPad Prism version 9.4.1 for Windows 64-bit, GraphPad Software, San Diego, California USA, www.graphpad.com. For Figures and Diagrams All Diagrams and figures in this thesis were "Created with BioRender.com" under an academic subscription.