Elyra ps 1 confocal microscope

The cells were fixed with 4% paraformaldehyde for 20 min at room temperature. Then they were washed three times with PBS 1x for 5 min. Next, the cells were permeabilized with 0.3% Triton diluted in 1x PBS for 15 min. Subsequently, blocking was done using a 5% BSA solution diluted in PBS 1x for 60 min. We then placed the primary antibody in question in PBS-BSA 3% at 1:100 (LMNA, cat. MA3-1,000, ThermoFisher scientific) for Lamin A/C; and 1:200 (Troponin T, cat. MS-295-P1, ThermoFisher scientific) for Troponin T, cardiac isoform, dilutions, and incubated overnight at 4°C. The following day, we washed 3x with 1x PBS for 5 min. After the washes, we placed the secondary antibody Cy™3 AffiniPure Donkey Anti-Mouse IgG (H + L) (cat. 715150, Jackson ImmunoResearch) diluted in PBS-BSA 3% at a 1:400 ratio for 2 h at room temperature. After incubation, we washed 3x with 1x PBS for 5 min. Finally, we placed DAPI (cat. D9542, Sigma-Aldrich) for 5 min and washed 3x times with PBS 1x for 5 min. The coverslips were sealed using 13 µL of Fluoroumont (ThermoFisher scientific). Images were taken with the ×100 objective on the Elyra PS.1 confocal microscope (Carl Zeiss), ZEN 2012 SP5 software (Carl Zeiss), at the Advanced Microscopy Unit (UMA) of the National Center for Structural Biology and Bioimaging (UFRJ, Rio de Janeiro).

Immunohistochemistry was carried out similarly to previously published protocols [26 (link),33 (link)]. Fixed hemispheres were mounted on a Leica VT1000 S Vibratome and sagittally sectioned into 50 μm slices. Primary antibodies (serine-129 phospho-alpha-synuclein: 1:667, 81A, BioLegend, San Diego, CA, USA; GFP: 1:3000, ab6556, Abcam, Cambridge, UK; actin: 1:100, A0483, Sigma-Aldrich, St. Louis, MO, USA) were incubated with shaking in the dark overnight at 4 °C. Tissue was washed in PBS five times for 30 min each time. Secondary antibodies, goat anti-mouse or goat anti-rabbit Alexa Fluor 647 (Invitrogen, Waltham, MA, USA), were diluted to 1:2000 and subjected to incubated shaking in the dark overnight at 4 °C. Tissue was washed in PBS five times for 30 min each time at room temperature. X-34 staining was performed using standard techniques and was provided as a gift from William Klunk (University of Pittsburgh, Pittsburgh, PA, USA) [35 (link)]. Phalloidin staining was performed using the manufacturer’s protocols (Alexa Fluor 555 Phalloidin, Fisher Scientific, Waltham, MA, USA). For confocal imaging, images were acquired on a Zeiss Elyra PS.1 confocal microscope with a Plan-Apochromat 63×/1.40 oil objective.

Spleens were frozen in Tissue‐Tek OCT Compound (VWR) and cryosectioned. Sections were fixed with 4% PFA for 10 min, washed three times in PBS, blocked 45 min with 10% goat serum in PBS, and stained 1 h with 100‐fold diluted antibodies (Pacific blue anti‐mouse B220, Alexa Fluor 488 anti‐mouse CD45.2, Alexa Fluor 594 anti‐mouse CD3 and Alexa Fluor 647 anti‐mouse GL7). After washing three times with PBS, stained sections were mounted with ProLong Diamond Antifade Mountant (Thermo Fisher). Images were acquired on a Zeiss Elyra PS.1 confocal microscope and analyzed in ImageJ 2.0.0.