Lsm 510 upright confocal microscope

Mineral deposition was evaluated by intraperitoneal injections of calcein (Sigma # C0875; 2.5 mg/kg body weight) and alizarin complexone (Sigma # a3882; 7.5 mg/kg) into pregnant females and, postnatally, into cubs. Two mice were used for each injection regime. Prenatally harvested limbs were fixated overnight in 4% PFA/PBS, dehydrated to 100% ethanol, embedded in paraffin and sectioned at a thickness of 7 μm. Postnatally harvested limbs were fixated 24 h in 4% PFA/PBS and gradually dehydrated from 70% ethanol to 100% ethanol twice for 48 h each time. Then, samples were infiltrated and embedded in JB-4 Embedding Kit (Electron Microscopy Science #14270–00) and sectioned longitudinally at a thickness of 7 μm. Fluorescence was visualized by confocal microscopy.
Confocal imaging was performed using a Zeiss LSM 510 upright confocal microscope (Carl Zeiss, Jena, Germany) with an EC Plan-Neofluar 10x/0.3 objective, NA 1.0. Calcein fluorochrome was excited with a 488 nm argon laser and alizarin with 561 nm argon laser. Following imaging, all images of the same section were stitched using Microsoft Image Composite Editor (version 1.4.4.0). Contrast was increased by using the “auto contrast” tool of Google Picasa (version 3.9.137) and Matlab’s “imadjust” function.

The biofilms were observed by confocal laser scanning microscopy (CLSM)^{52 (link)}. Briefly, all strains were grown in glass bottom cell culture dishes. After 24 h of incubation at 37 °C, the resulting biofilms were washed and stained with 25 µg/ml concanavalin A-Alexa Fluor 594 conjugate (Invitrogen) at 37 °C for 1 h. CLSM was performed with a Zeiss LSM 510 upright confocal microscope (Carl Zeiss) using a Zeiss Achroplan ×40, 0.8-W objective, and a He-Ne laser with an excitation wavelength of 543 nm. Moreover, biofilm activity was assessed with an XTT reduction assay according to previously described protocols^{53 (link),54 (link)}.

RPE samples were incubated with 10 μg/ml Dil-conjugated AcLDL (Molecular Probes). Dil-AcLDL was added only to the upper chamber of the transwell membrane insert. After 2, 4, 6, and 12 h, samples were washed in DPBS to remove any excess Dil-AcLDL still present in the medium. RPE monolayers or monolayer fragments were gently detached from the transwell membrane with the aid of a P10 pipette tip. RPE monolayers or monolayer fragments were mounted on Poly-l-lysine-coated microscope slides (Tekdon) prior to imaging.
To look for evidence of POS phagocytosis in Dil-AcLDL-positive cells, hESC-derived RPE were first subjected to a 12-h incubation with medium containing Dil-AcLDL followed by a DPBS wash and an 8-h incubation with POS. After completion of incubation with POS, cells were washed to release any surface-bound POS. Images were acquired using a Zeiss LSM 510 upright confocal microscope using a Plan-Apochromat 10x/0.45 or 20x/0.8 objective. FITC and Alexa-568 fluorophores were excited with a 488-nm (30 mW) and 543-nm (1 mW) laser line and collected with a FITC and TRITC filter set. Image acquisition and processing were performed in AxioVision software (Zeiss).

CFM was conducted using a Zeiss LSM510 Upright Confocal Microscope of samples grown directly on clean glass substrates, under oil immersion where required. Low-magnification images were obtained to demonstrate dye distribution across the population of composites, and higher-magnification z-stacks were performed on at least three individual crystals per sample. Lasers and filters were selected based on their suitability to the excitation and emission maxima of the dye. Intensities of different lasers were required due to different dye quantum yields and overall final mol% in composites. Image rendering and analysis was conducted in ImageJ.