Zen acquisition software

After fixation and immunostaining, images were acquired with an inverted laser scanning confocal microscope LSM700 (Zeiss, Germany), with a 63X objective (zoom 1.0). Images were acquired using the ZEN acquisition software (Zeiss). In all experiments, we acquired Z-stacks covering the whole volume of cells. For the experiments where integrated density was measured in control and α-syn treated cells (LysoTracker DR, Magic Red CathB, DQ-BSA assays, and evaluation of LAMP1 at the PM in non-permeabilized condition), all settings (including the laser power and exposure line time) were kept identical. In some experiments, line averaging were also used in order to improve the signal-to-noise ratio.

HeLa Kyoto cells expressing MYH9-EGFP and Lifeact-mCherry, MYH9-EGFP or Actin-EGFP were prepared for experiments in glass bottom dishes, chemically arrested in mitosis in the presence of 2 µM STC and mounted on an inverted light microscope (AxioObserver.Z1, Zeiss) equipped with a laser scanning microscope (LSM 700, Zeiss) using a dish heater (JPK Instruments) at a constant temperature of 37 °C on a manual or automated precision stage (JPK Instruments). Imaging was carried out using a ×63 water-immersion objective (LCI Plan-Neofluar 63x/1.3 Imm Corr DIC M27) and ZEN acquisition software (Zeiss). Using modified (‘wedged’) cantilevers compensating for the 10° tilt intrinsic to our AFM setup and thus preventing cells from sliding towards the cantilever base^{5 (link), 19 (link)}, the cantilever was brought into contact with the glass surface close to the cell of interest, retracted by at least 22 µm, positioned over the cell and subsequently lowered with a constant speed of 0.5 µm s⁻¹ to a predefined set height (10 or 12 µm). Images at the cellular midplane were recorded and cortical enrichment of myosin II and actin were analyzed by comparing the average fluorescence intensity of MYH9-EGFP, Lifeact-mCherry or Actin-EGFP at the cell edge to that in the cytoplasm using a custom Igor image analysis macro (Igor Pro)^{11 (link)}.

Paraffin-embedded lymph node sections were deparaffinized, incubated in citrate buffer (10 mM sodium citrate) for antigen retrieval, and permeabilized in TTBS (10 mM Tris, 150 mM NaCl, 0,05% Tween20). 0.1% Sudan black B (Sigma-Aldrich/Merck, Darmstadt, Germany) in 70% ethanol was applied as described before to reduce autofluorescence [119 (link)]. After washing with TTBS, sections were blocked with 5% normal horse serum (Invitrogen/Thermo Fisher Scientific, Waltham, MA, USA) in TTBS. Primary antibodies listed in Supplementary Table S8 were diluted in TTBS, administered to the slides, and incubated overnight at 4 °C. The next day, slides were washed with TTBS and secondary antibodies were added to incubate for 45 min at room temperature in the dark. Slides were washed and mounted using mounting medium with DAPI (SouthernBiotech, Birmingham, AL, USA) to stain nuclei. Images were acquired using a Zeiss cell observer widefield microscope and ZEN acquisition software (both Carl Zeiss, Oberkochen, Germany).

Images of stained sections were acquired using a Zeiss LSM510 confocal scanning microscope (Carl Zeiss, GER) as previously described (Biancardi et al. 2010; Iddings et al. 2015). Images from consecutive optical focal planes (1 μm interval), were taken using a 25× or 40× oil immersion objective, and a projection image of the sections was generated. For NMDAR (n = 6, per group), FITC dextran (n = 13, per group), Iba1 (n = 13, per group) and GFAP (n = 13, per group) 30 μm consecutives images were acquired at 40×. Additionally, for FITC dextran (n = 10, per group) 50 μm consecutive images were acquired at 25×. Images from both hemispheres of WKY and SHR groups were acquired by Zen acquisition software (Carl Zeiss Microscopy, Oberkochen, Germany) and digitized with identical acquisition settings for further comparison.