Cells on glass coverslips were maintained in 6-well dishes, fixed in 4% paraformaldehyde in PBS for 20 min, permeabilized with 0.25% Triton X-100 in PBS for 5 min, and stained overnight at 4°C with primary antibodies. The next day, samples were washed and stained with secondary antibodies conjugated to Alexa Fluor fluorochromes (Thermo Fisher Scientific) and Hoechst (Thermo Fisher Scientific).
Alexa fluor fluorochromes
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor fluorochromes are a series of synthetic fluorescent dyes developed by Thermo Fisher Scientific. These dyes are designed to provide bright, photostable fluorescence across a wide range of wavelengths. They are commonly used in various biomedical research applications, such as flow cytometry, microscopy, and immunoassays.
Automatically generated - may contain errors
2 protocols using alexa fluor fluorochromes
1
Immunofluorescence Staining of Cells
2
Immunohistochemistry and Immunoblotting of Aortic Lysates
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For immunohistochemistry, aortas from 6-wk-old C57BL/6 mice were embedded in cryoblock medium (VWR International) on dry ice. Cryosections were incubated with anti-LOX (Abcam, Cambridge, MA, USA) or anti-LOXL2 (Santa Cruz Biotechnology, Dallas, TX, USA) before fixation with acetone and further processing with secondary antibodies coupled to Alexa Fluor fluorochromes (Thermo Fisher Scientific) as previously described in Bignon et al. (9 (link)).
Frozen aortas were pulverized on dry ice before lysis in 25 mM Tris (pH 7.5), 100 mM NaCl, 5 mM EDTA, and 1% Triton X-100 containing Protease Inhibitor Cocktail (MilliporeSigma). Samples were centrifuged for 30 min at 15,000 g, and pellets were solubilized in Laemmli buffer. Proteins of both lysates were separated by SDS-PAGE and electrotransfered to PVDF membranes for immunodetection of LOX, LOXL2 (Abnova), fibronectin (Merck), and actin (Abcam).
Frozen aortas were pulverized on dry ice before lysis in 25 mM Tris (pH 7.5), 100 mM NaCl, 5 mM EDTA, and 1% Triton X-100 containing Protease Inhibitor Cocktail (MilliporeSigma). Samples were centrifuged for 30 min at 15,000 g, and pellets were solubilized in Laemmli buffer. Proteins of both lysates were separated by SDS-PAGE and electrotransfered to PVDF membranes for immunodetection of LOX, LOXL2 (Abnova), fibronectin (Merck), and actin (Abcam).
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