Leica tcs sp

Dextran coupled with FITC (FITC-dextran, Sigma-Aldrich) was chosen to assess the alterations of arterial endothelial permeability of collateral vessels. Rats were injected via the tail artery with 0.25 ml of 5% FITC-dextran per 100 g body weight 2 hours before they were killed. The musculus gracilis was removed and cryosections were cut 5-μm. Then immunofluorescent staining for CD31 (endothelial marker, detected by cy3 fluorescein) was performed. The sections were viewed with a Leica confocal microscope (Leica TCS SP). The amount of FITC-dextran in the musculus gracilis was determined with confocal microscopy.

In all cases, no cross-reactivity was detected when one or two of the primary antibodies were omitted.
Sections were analyzed either with a Leica TCS SP (Leica, Wetzlar Germany) or a Zeiss LSM 5 Pascal (Zeiss, Jena, Germany) laser-scanning microscopes using sequential acquisition of separate wavelength channels to avoid fluorescence cross talk. Single confocal optical slices and z-stacks of 10–25 confocal optical slices (2,048 × 2,048 pixels) spaced 200–300 nm were acquired with a 100X objective (oil immersion, 1.4 numerical aperture), a numerical zoom 2 (37.8 nm/pixel) or zoom 8 (9.4 nm/pixel) and with the pinhole set at 1 Airy unit. Images were displayed with the NIH ImageJ software (Abràmoff et al. 2004 ).

Dynamic light scattering (DLS) and ζ-potential determinations were performed on a ZetaSizer Nano ZS (Malvern Instruments). The morphology and size of the simples were investigated by transmission electron microscopy (TEM) on a JEM-1011 microscope (JEOL, Japan) and atomic force microscope (AFM) images were collected by FASTSCANBIO (Bruker) in a tapping mode. UV-vis absorption spectra were measured in UV-vis spectrometer (UV-2900, Shimadzu, Japan) equipped with a 1-mm quartz cell. Confocal images were collected on confocal laser scanning microscope (CLSM, Leica TCS SP) using a 60× oil-immersion objective.

Confocal imaging was performed on either a Leica TCS SP or a Leica TCS SP5 II (Leica GmbH, Heidelberg, Germany). Cells seeded on coverslips were clamped into custom-made aluminum rings for imaging 16 h post transfection covered with 500 µL of phosphate buffer saline (PBS ) medium (8 g/L sodium chloride, 0.2 g/L potassium chloride, 1.42 g/L disodium hydrogen phosphate, 0.24 g/L potassium hydrogen phosphate; pH was adjusted with 1 M sodium hydroxide up to 7.4). Cells were imaged with either PL APO 100 × 1.40 OIL UV or HCX PL APO 63 × 1.20 W CORR UV objectives. Dyes or fluorescent proteins were excited with an argon (488 nm), krypton (568 nm) or helium-neon (632 nm) laser.

At day 7 post-surgery, The animals were sacrificed under overdose of anesthesia with sodium pentobarbital (80 mg/kg, intraperitoneally). Gracilis muscles were removed for exepiments. By our previous experience, this muscle contains 1–2 collateral vessels constantly during arteriogenesis. A total of 48 vessels were investigated. All samples were immediately frozen in liquid nitrogen, embedded in tissue processing medium (O.C.T) and stored at -80°C till further use. Cryosections were cut 5-μm thick, fixed in 4% paraformaldehyde, then pre-incubated in 0.2% BSA-C (Aurion Co.) and thereafter incubated with the primary antibodies (Table 1). Incubation of second antibodies (Table 1) at a concentration of 1:200 was followed by Cy2 conjugated Streptavidin (Biotrend). The nuclei were stained with TOTO3 (Molecular Probes). The sections were coverslipped and viewed with a Leica confocal microscope (Leica TCS SP). Further documentation and image analysis were carried out using a Silicon Graphics Octane workstation (Silicon Graphics) and three-dimensional multichannel image processing software (Bitplane).
Immunostaining for cultured cells was performed following a similar protocol as described above, except for primary antibody incubation that was conducted at 4°C overnight. The nuclei were stained with 7-aminoactinomycin D.