Lsm 980 elyra 7

FRAP experiments were performed on ZEISS LSM 980 Elyra 7 super-resolution microscope equipped with a high-resolution monochrome cooled AxioCamMRm Rev. 3 FireWire(D) camera, using a 63x oil-immersion objective (Numerical aperture 1.4). Alexa-488-labeled α-Syn and PrP (~ 1 %) were used for FRAP experiments. A region of interest (ROI) with a radius of 0.5 μm was bleached using a 488-nm laser. The recovery of the bleached spots was recorded (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint this version posted September 6, 2021. ; https://doi.org/10.1101/2021.09.05.459019 doi: bioRxiv preprint 23 using ZEN Pro 2011(ZEISS) software provided with the instrument. The fluorescence recovery curves were background corrected, normalized, and plotted using Origin.

All the imaging experiments were performed at room temperature on ZEISS LSM 980 Elyra 7 super-resolution microscope equipped with a high-resolution monochrome cooled AxioCamMRm Rev. 3 FireWire(D) camera, using a 63x oil-immersion objective (Numerical aperture 1.4). For visualizing droplets of PrP-α-Syn and PrP-α-Syn-RNA complexes, 1% of unlabeled proteins were doped with labeled proteins, and the samples were placed in Labtek chambers. Alexa-488-labeled protein was imaged using a 488-nm laser diode (11.9 mW), and Alexa-594-labeled protein was imaged using an excitation source at 590-nm. The ThT positive fibrils were imaged using a 402-nm excitation source. Images were processed and analyzed using ImageJ (NIH, Bethesda, USA). Concentrations in the dense phase and the light phase were estimated using a previously defined protocol. 75 Calibration plots were generated from fluorescence intensities of the dispersed phase of Alexa-488-labeled α-Syn and Alexa-594labeled PrP at different concentrations. The confocal images of droplets formed using Alexa-488-labeled PrP, and Alexa-594-labeled α-Syn (labeled protein: 0.1%) were then analyzed using ImageJ software to get an approximate estimation of protein concentration inside droplets. The Csat was estimated using a similar method (labeled protein: 2%) and was verified using SDS-PAGE analysis as described above.

For labeling, purified FUS was concentrated using a 50 kDa MWCO amicon filter, and incubated with 0.3 mM tris(2-carboxyethyl)phosphine (TCEP) for 30 minutes on ice following Fluorescence recovery after photobleaching (FRAP) measurements FRAP experiments for droplets with and without Ag IMNPs were performed on ZEISS LSM 980 Elyra 7 super-resolution microscope using a 63x oil-immersion objective (Numerical aperture 1.4). All the FRAP experiments were performed using 200 nM (1%) of Alexa488labeled protein. The recovery of the chosen region of interest (ROI) after photobleaching using a 488-nm laser was then recorded using ZEN Pro 2011 (ZEISS) software provided with the instrument. The fluorescence recovery curves were then normalized and plotted after background correction using Origin.