Lsm 5 exciter confocal microscope

Selaginella root tips were first fixed in 50% methanol and 10% acetic acid and after clearing subjected to a modified pseudo-Schiff propidium iodide staining as described previously (Truernit et al., 2008 (link)). Analysis was done with a Zeiss LSM5 Exciter confocal microscope with an argon ion laser at 488 nm as the excitation source and a detection filter at 505 nm. For all samples, z-stacks were taken to ensure the possible detection of meristematic regions in different planes.

Biofilms of S. pseudintermedius DSM21284 were pre-formed on μ-Plate 96 well uncoated microtitre plates (Ibidi, Germany) suited to confocal microscopy applications. Following peptide treatment, biofilms were rinsed once with PBS and stained by using a Live/Dead BacLight viability kit (Molecular Probes). 100 ml of the solution containing SYTO 9 and propidium iodide mixed in a ratio of 1:1 was added to the biofilm. The films were incubated at room temperature for 15 min in the dark. After incubation, residual stain was removed. The images were observed using a Zeiss LSM 5 exciter confocal microscope with a Plan-Apochromat 63x/1.40 Oil DIC M27 lens and images were acquired using the Zen 2008 SP2 software. Sample were excited using laser light at 488nm with emission light filtered with a bandpass filter at 505–530 nm for Syto 9 and a longpass filter at 650 nm for propidium iodide (PI). Images were acquired using two separate confocal channel (one for Syto 9 and one for PI) with pinhole adjusted to 1 (confocal pinhole) at 1556 × 1556 pixels.

Cells were plated onto submerged glass coverslips and seeded in 6-well plates for 24 h. After drug treatment, cells were fixed with methanol for 2 min and permeabilized with 0.1% Triton X-100 in DPBS (Dulbecco’s phosphate-buffered saline) for 10 min at room temperature (RT). Coverslips were blocked with 1% bovine serum albumin (BSA) in DPBS with Tween 20 (DPBS-T), and cells were incubated with antibodies against GR overnight at 4°C. After washing with DPBS-T and incubation with Alexa 488-conjugated secondary antibodies for 90 min at RT in the dark, nuclei were counterstained with 0.2 μg/ml DAPI. Images were obtained using a Carl Zeiss LSM 5 exciter confocal microscope (Carl Zeiss AG).

COS-7 cells expressing CDK5 and p35 or its mutants were cultured on cover slips and fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 30 min at room temperature, which was followed by incubation with an anti-myc (1:1000) antibody in PBS containing 5% normal goat serum and 0.1% Triton X-100 for 1 h and a second incubation with a secondary antibody conjugated to Alexa Fluor 488 (1:500) [20 (link)]. Nuclei were stained with DAPI. Cells were examined under a LSM 5 EXCITER confocal microscope (Carl Zeiss) or a fluorescence microscope (BZ-X700; Keyence, Osaka, Japan).

For the observation of the initial cell morphology of hMSCs seeded in composite scaffolds, the cells were fixed in 4% paraformaldehyde at room temperature. After washing in PBS, to block unspecific protein interactions, the samples were put in blocking solution (0.3% Triton X-100, 1% BSA, 10% donkey serum). Then, the samples were incubated with WGA-Alexa Fluor conjugate (Life Technologies) at 37°C for 1 h. Also, to observe the expression of osteopontin, one of the bone differentiation proteins, 10 μg mL⁻¹ osteopontin primary antibody (Abcam) was incubated overnight at 4°C. After washing, FITC-conjugated secondary antibody was incubated for 1 h at room temperature. Then, the samples were washed for nuclear labeling in the cells using mounting media (within DAPI, VECTOR) and observed using an LSM 5 Exciter confocal microscope (Carl Zeiss Microscopy GmbH, Olympus, Japan).