Anti lox

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).

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).