Thick 100-µm slices of bovine cortical bone were sterilized with 70% ethanol, rinsed with sterile deionized water followed by overnight equilibration in DMEM media. Bone slices were then overlaid with 2.85 × 105 RAW 264.7 cells in the presence of 35 ng/mL RANKL and 25 µM KGP94. Four days later, the osteoclasts were scraped off the bone slices using a cotton swab. Bone slices were then rinsed in PBS and resorption pits were stained using 1% toluidine blue solution in 0.5% tetraborate. Resorption pits were imaged using a Zeiss Axioplane 2 imaging system and pit area was quantified using ImageJ software by calculating the area of toluidine positive region for each field of view at 10 × magnification. Four random fields were captured from each bone slice and four bone slices were tested under each experimental condition. Extent of bone resorption is expressed in terms of percent area of bone that was positively stained.
Axioplane 2 imaging system
Manufactured by Zeiss
The Axioplane 2 is an imaging system designed for high-resolution microscopy. It features a stable and precise optical path, enabling consistent and reliable image capture. The system is equipped with advanced optics and illumination components to support a variety of imaging techniques, including bright-field, dark-field, and fluorescence microscopy.
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Lab products found in correlation
2 protocols using axioplane 2 imaging system
1
Quantifying Osteoclast-Mediated Bovine Bone Resorption
2
Trophoblast Chromosome Enumeration by FISH
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Isolated trophoblast were centrifuged and incubated with 0.5% KCL and incubated at 37°C for 20 minutes. Subsequently, cells were treated with ice-cold fixative (one part acetic acid and three parts methanol) and incubated for 10 minutes at -20°C. Fixed cells were air-dried on glass slides at 42°C for 15 minutes and treated with pepsin (350 μl 0.5% Pepsin with 1 ml 1N HCl ad 100 ml H2O) for 15 min at 37°C. Slides were then washed in PBS and PBS/20mM MgCl2 each 5 minutes at room temperature. Subsequent to dehydration in a 10% formaldehyde solution, cells were incubated with a “ready to use” solution containing centromer specific probes for X- and Y-chromosomes (DXZ1 (green) and DYZ3 (red) (Leica Biosystems). Hybridization was performed using a ThermoBrite system (Leica) for 16 h at 37°C after 5 minutes denaturation at 75°C. Afterwards, cells were washed for two minutes with 0.4x SSC with 0.3% NP40 followed by a 1 minute wash step with 2x SSC with 0.1% NP40. Slides were air-dried and covered with Vectashield Mounting Medium containing DAPI (Vector Laboratories Burlingame, USA). FISH signals were detected using an Axioplane2 imaging system (Zeiss) equipped with “MetaSystems Isis” software version 5.3.18.
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