Lsm 880 airy nlo

To observe the subcellular distribution of vinculin, cells were washed in PBS after 3, 6 and 24 h of cultivation and then were fixed in 10% paraformaldehyde for 30 min at room temperature. The cells were then washed three times in PBS, after which nonspecific binding was blocked with 1% fetal bovine serum (FBS) for 30 min at room temperature.
To observe actin filaments, the cells were incubated for 30 min at room temperature with FITC-conjugated phalloidin (1:100 dilution, Invitrogen, Waltham, MA, USA) or were incubated overnight at 4 °C with a primary rabbit anti-vinculin antibody (1:100 dilution, Invitrogen, Waltham, MA, USA). After three additional washes in PBS, the samples were incubated with a secondary antibody, Alexa fluor 488 goat anti-rabbit immunoglobulin G (IgG) (1:100 dilution, Invitrogen, Waltham, MA, USA), which was detected as green fluorescence for 30 min at room temperature. After five washes in PBS, a micro cover glass was placed over each sample overnight for 4 °C with ProLong with DAPI (Invitrogen, Waltham, MA, USA).
These samples were observed using a confocal laser scanning microscope (LSM 880 Airy NLO) with software (Zen, Carl Zeiss, Oberkochen, Germany).

Second harmonic generation (SHG) images were obtained using a multiphoton confocal microscopy system (LSM 880 Airy NLO, Carl Zeiss, Oberkochen, Germany) with excitation laser (Chameleon Vision II, wavelengths: 680–1080 nm; repetition rate: 80 MHz pulse width: 140 fs, Coherent Inc., Santa Clara, CA, USA) and objective lens (Plan-Apochromat 10x/0.8 M27, Carl Zeiss, Oberkochen, Germany). The excitation wavelength for observing collagen fibers was 880 nm. From the image obtained, a region of 200 μm × 200 μm square at the implant neck (A and F) was extracted as the region of interest. High-precision image analysis software (Imaris8.4, Bitplane AG, Zürich, Switzerland) was used to trace and measure the angles of the collagen fiber bundles.

Second harmonic generation (SHG) images were obtained using a multiphoton confocal microscopy system (LSM 880 Airy NLO; Carl Zeiss, Oberkochen, Germany) with an excitation laser (Chameleon Vision II, wavelengths: 680-1080 nm; repetition rate: 80 MHz; pulse width: 140 fs; Coherent Inc. Apochromat 10×/0.8 M27; Carl Zeiss, Oberkochen, Germany). The excitation wavelength for observation of collagen fibers was 880 nm. Software (ZEN, Carl Zeiss, Oberkochen, Germany) was used for image acquisition. After image acquisition, the collagen fiber bundles in the calcified area of interest were traced using Imaris 8.4 (Bitplane AG, Switzerland), and the fiber bundle diameters were measured.