Fluoview fv1200 confocal laser scanning biological microscope

HL-1 cells were cultured as above, and they were treated with or without aggLDL (100 μg/mL) for 8 h and then stimulated or not with 100 nM of insulin for 30 min together with 80 μΜ of 2-Deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl) amino]-D-glucose (2-NBDG solution; Sigma-Aldrich, St. Louis, MO, USA). The 2-NBDG is a glucose fluorescent analog that is not metabolized and displays emission maxima of 540 nm. After stimulus, cells were washed with PBS 1X, fixed with 4% of paraformaldehyde, quenched with 50 mM NH₄Cl, permeabilized for 30 min with 0.5% (v/v) saponin, blocked with 2% BSA and incubated with Hoechst 33,258 colorant (1/2000) for 1 h. Finally, cells were mounted on glass slides with Mowiol 4–88 reagent. Fluorescent images were obtained with an Olympus FluoView FV1200 confocal laser scanning biological microscope (Olympus, NY, USA). Whole cells were scanned and optical sections were obtained in 0.25-μm steps perpendicular to the z-axis, with images being processed using the FV10-ASW Viewer 3.1 (Olympus, NY, USA) and the total fluorescence in the whole cell area was quantified by ImageJ software. For microscope quantification of the co-localization level, a JACoP plug-in from ImageJ software (NIH, Bethesda, MD, USA) was used.

The procedures were followed as previously described^{29 (link),42 (link)}. Briefly, MIO-M1 cells were cultured as above on cover glass. After 30 min of IGF-1 stimulus the cells were washed with PBS 1×, fixed with 4% PFA, quenched with 50 mM NH₄Cl, permeabilized for 30 min with 0.5% (v/v) saponin, blocked with 2% bovine serum albumin (BSA) and incubated with primary antibodies mouse anti-GLUT1 (1/100) and rabbit anti-LRP1 (1/200) for 1 h, and revealed with a secondary antibody conjugated with Alexa Fluor 594 or 488 (1/800) and Hoechst colorant (1/2000) for 1 h. Finally, the cells were mounted on glass slides with Mowiol 4–88 reagent from Calbiochem (Merck KGaA, Darmstadt, Germany). For co-localization analyses, fluorescent images were obtained with an Olympus FluoView FV1200 confocal laser scanning biological microscope (Olympus, NY). Whole cells were scanned and optical sections were obtained in 0.25-μm steps perpendicular to the z-axis, with images being processed using the FV10-ASW Viewer 3.1 (Olympus) and quantified by ImageJ software (NIH, Bethesda, Maryland)^{48 (link)}.