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 previously77 (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.
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 previously77 (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.
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