Tcs sp2 spectral confocal microscope

Liver cryosections (5μm) were fixed with 4% paraformaldehyde and blocked for 1 hour with mouse IgG blocking reagent (vector labs). After blocking with Donkey serum (5% in PBS-0.3%Tween), sections were incubated with 1/500 anti-pericentrin antibody (Abcam) overnight. Secondary 1/500 Anti-rabbit Cy3 (Jackson immunoresearch, stratech) and 1/500 Alexafluor 488 –phalloidin (invitrogen) was then applied on the sections for 1-2hours. Sections were mounted with vectashield plus DAPI medium. Sections were viewed and analysed using a Leica TCS SP2 spectral confocal microscope and Leica confocal software (Leica, Heidelberg, Germany). Centrosomes numbers were counted in at least 150 nuclei of hepatocytes from each mouse for each genotype (N=3-5) at D4 and D6.

For fluorescent microscopy, J774.E cells were seeded in 24-well plates while for confocal microscopy the cells were seeded in Laboratory-Tek II 4-chamber cover glasses slides. When the cells reached ~ 80% confluence they were incubated with 80 nM NCs in 20 mM HEPES-buffered Hanks’ balanced saline solution (HBSS) for 1 hour at 37°C. The incubation media included a general fluid endocytosis marker tetramethylrhodamine B dextran (10,000 MW) at 50 μM and the nuclear dye DAPI at 5 μg/ml. The cells were then washed with PBS five times. NC uptake into live J774.E cells was examined under an inverted Olympus fluorescence microscope (model IX71S1-F3) at the magnification of 40x objective. Confocal microscopy was performed on a Leica TCS SP2 Spectral confocal microscope using the XYZ mode. A sequential image acquisition mode was used to avoid possible partial light spillover from one color to another. The middle cell sections of selected Z-stacks are shown in Fig. 8. Quantification of green fluorescence within individual cells and in a middle section of an acquired Z-stack was performed using Leica’s software, LAS AF, version 2.4.1.

ALP was purchased from Biomatik, NBD-Cl from TCI and amino acids from GL
Biochem, PP1 from Sigma-Aldrich, PTP1B from Abcam. All the solvents and chemical reagents
were used directly as received from the commercial sources without further purification.
All the products were purified with Water Delta600 HPLC system, equipped with an XTerra
C18 RP column and an in-line diode array UV detector. Rheological data were obtained on TA
ARES G2 rheometer with 25 mm cone plate, confocal microscopy images on Leica TCS SP2
spectral confocal microscope. Electron microscopy imaging was performed on a FEI Morgagni
268 TEM with a 1k CCD camera (GATAN, Inc., Pleasanton, CA, USA) or a 300 keV Tecnai F30
intermediate voltage TEM (FEI, Inc., Hillsboro, OR, USA) with a 4k CCD camera (GATAN).

Iron oxide nanoparticles with amphiphilic polymer coating and a monolayer of oleic acid were purchased from the Ocean NanoTech company. Transmission electron microscope images were collected by using Morgagni 268 microscope. Confocal images were taken with the Leica TCS SP2 Spectral Confocal Microscope. The cells were counted by a hemacytometer.