Alexa fluor 594 protein labeling kit

After sonication, α-syn PFFs (2 mg/mL) were labeled with the Alexa Fluor 594 protein labeling kit (Thermo Fisher, A10239) according to the manufacturer’s instructions. Time-lapse imaging was performed with a BZ-X710 camera (Keyence) equipped with an incubator (37 °C, 5% CO2), and images were acquired at defined positions every 10 min. The images were then converted to mp4 files. Immediately after time-lapse imaging, cells were fixed and subjected to immunostaining to validate the maturation status of the imaged cells (Additional file 5: Fig. S4B, C).

TTR protein and BSA were labeled with the Alexa Fluor 594 protein labeling kit (Thermo Fisher Scientific) following the manufacturer’s instructions. Briefly, 100 µL TTR proteins and BSA (0.1 µg/µL) were incubated with 4.7 µL Alexa Fluor 594 succinimidyl ester (12.2 nmole/µL) for 15 min at room temperature, and the conjugated reaction mixture was then purified with resin gel-spin filter. Labeled TTR proteins (0.2 µg) and BSA were added to the cells and detected by fluorescence microscope (Nikon, Tokyo, Japan).

RBD fragment of the Spike protein belonging to SARS-CoV-2 (Raybiotech, Peachtree Corners GA. Cat. 230-30162-1000) was labeled using Alexa Fluor 594 Protein Labeling Kit (ThermoFisher Scientific, Waltham, MA. Cat. A10239) following the manufacturer’s directions. Briefly, 1 mg of protein was dissolved in 0.1 M bicarbonate and then incubated with the Alexa Fluor 594 dye for one hour. The dye was washed using an Amicon-Ultra centrifugal filter cutoff 10 kDa (Merck, Millipore Carrigtwohill, CO. Cat. UFC201024). To assess the efficiency of the label, the protein was measured at 280 nm and 590 nm absorbance using NanoDrop One (ThermoFisher Scientific). There was a ratio of 0.4 mol of dye/mole of protein and a recovery of about 80%.

RBD fragment of the Spike protein belonging to SARS-CoV-2 (Raybiotech, Peachtree Corners GA. Cat. 230-30162-1000) was labeled using Alexa Fluor™ 594 Protein Labeling Kit (ThermoFisher, Waltham, MA. Cat. A10239) following the manufacturer’s directions. Briefly, 1 mg of protein was dissolved in 0.1 M bicarbonate and then incubated with the Alexa Fluor 594 dye for one hour. The dye was washed using an Amicon-Ultra centrifugal filter cutoff 10KDa (Merck, Millipore Carrigtwohill, CO. Cat. UFC201024). To assess the efficiency of the label, the protein was measured at 280 nm and 590 nm absorbance using NanoDrop One (Thermo Scientific). There was a ratio of 0.4 moles of dye/mole of protein and a recovery of about 80%.

Anti-ricin Ab was conjugated (1 mg) with the Alexa Fluor 594 protein labeling kit (Molecular Probes, Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions.
For immunolocalization experiments, HEK293 cells or a single cell suspension (SCS) from mice lungs were seeded on #1 glass cover slips in 24-well dishes and exposed to ricin (100 ng/ml). Cells were fixed with 4% paraformaldehyde (PFA, Gadot, Israel) for 10 min at 4 °C, washed three times with PBS, and placed for 1 hour in a blocking solution (10% normal goat serum (NGS)) in PBS containing 0.05% Tween-20 (P5927, Sigma-Aldrich). Cells were incubated in a 1:500 dilution of rabbit anti-LRP1 Alexa Fluor 488 (Abcam, Cambridge, MA, USA) and anti-ricin Alexa Fluor 594 in an antibody cocktail solution (50% blocking solution/0.05% Tween-20/PBS) for 24 hours at 4 °C. Cover slips were processed as described previously^{77 (link)} and imaged in a sequential manner using an LSM 710 confocal scanning microscope (Zeiss, Jena, Germany) equipped with following lasers: argon multiline: 458/488/514 nm); diode: 405 nm; DPSS: 561 nm; and heliumneon: 633 nm. The percent of colocalization was quantified using Zen software (version 2.1, 2008; Zeiss). Image parameters: Scan mode-plane; Dimensions- X:1691 Y:1127 8-bit; Average-8; Pixel dewl-1.27 µs.