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Alexa fluor 569

Manufactured by Thermo Fisher Scientific

Alexa Fluor 569 is a fluorescent dye produced by Thermo Fisher Scientific. It is designed to emit light in the orange-red region of the visible spectrum, with an excitation maximum at 578 nm and an emission maximum at 603 nm. This dye can be used in a variety of biological and chemical applications, including fluorescence microscopy, flow cytometry, and protein labeling.

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2 protocols using alexa fluor 569

1

Mitochondria Localization and Immunostaining

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The 250,000 cells were plated in glass coverslips in 6-well plates and allowed to attach for 24h at 37°C (5% CO2). The cells were incubated in 1:5000 concentration of Mitotracker Deep Red (Invitrogen) for 15 minutes, washed with prewarmed media, then fixed and permeabilized in 100% methanol for 5 minutes at 4°C. Next, the cells were blocked with 5% BSA in PBS-TT (0.5% Tween and 0.1% Triton) for 1h and immunostained for mGPDH using monoclonal antibody anti-mGPDH (Abcam, Cambridge, MA) for 2h then secondary antibodies conjugated with Alexa Fluor 488 or Alexa Fluor 569 for 1h (Life Technologies, Thermo Fisher Scientific). DNA was stained with DAPI (Vector Laboratories, Burlingame, CA). Fluorescence images were detected by confocal microscope NLO 710 Zeiss, and collected using Carl Zeiss Zen Software (Zeiss, Germany).
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2

Immunocytochemistry Protocol for Quantifying Mitotic Lox Levels

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Forty-eight hours after transfection with siControl or siLOX, 250,000 cells were plated in glass coverslips in 6-well plates and allowed to attach for 24 hr at 37°C and in a 5% CO2 atmosphere. Cells were then fixed in 4% paraformaldehyde (PFA) for 15 min. After permeabilization with 70% ethanol, cells were blocked with 5% BSA in PBS-TT (0.5% Tween and 0.1% Triton) for 1 hr at RT. The cells were immunostained for LOX using monoclonal antibody anti-LOX (Abcam), anti-tubulin (Cell Signaling Technology or DSHB Univ. Iowa), and anti-pH3(Ser10) (Cell Signaling Technology). Then the cells were incubated with appropriate secondary antibodies conjugated with Alexa Fluor 488 or Alexa Fluor 569 for 1 hr (Life Technologies, Thermo Fisher Scientific). DNA was stained with DAPI (Vector Laboratories, Burlingame, CA). For the immunofluorescence staining of the mitotic fractions the samples were fixed in PFA 4%, washed and reconstituted in PBS before overnight incubation on poly-lysine coated cover slips (BD Biosciences) at 4°C. The samples were then stained with Hoechst 33342 (Sigma Aldrich) to detect DNA and with tubulin and LOX antibodies as described above. Fluorescence images were detected by confocal microscope NLO 710 Zeiss, and images were collected using Carl Zeiss Zen Software (Zeiss, Germany).
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