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Rabbit bones were fixed in 10% phosphate-buffered formalin for 24 hours.
The rabbit bones were fixed in 10% phosphate-buffered formalin, and decalcified with 10% EDTA.
Oligonucleotides were designed as follows: -GAPDH 5'-CAAAGTGGAGATTGTTGCCAT-3'-CACCACCTTCTTGATGTCATC-5C-Cathepsinpsin K 5'-TTAATTTGGGAGAAAAACCT-3'-AGCCGCCTCCACAGCCATAAT-5T-Calcitoninonin receptor 5'-TGGTGGAGGTTGTGCCCAATGGAGA-3'-CTCGTGGGTTTGCCTCATCTTGGTC-5C-5' Bones were fixed in PBS plus 4% paraformaldehyde over night at 4°C and then stored in 70% ethanol.
Following ex vivo bioluminescence imaging, selected organs were fixed in 10% neutral buffered formalin and long bones were fixed in 4% paraformaldehyde in phosphate buffer for 1 week followed by decalcification in 14% EDTA (with 14 consecutive changes) at 4°C.
Bones were fixed in 10% buffered formalin prior to treatment with acid for removal of calcium.
For TRAP histochemistry, femur bones were fixed in 4% formaldehyde and decalcified using EDTA solution.
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Bones or OS were fixed in 4% paraformaldehyde and decalcified for 14 21 days in 14% EDTA.
Five AC samples (ages 50 to 60 years; mean age 56 years) from the medial and lateral compartments (femoral condyles and tibial plateaus) with a 'normal' macroscopic appearance were harvested and full thickness sections excluding subchondral bone were fixed in formalin and processed to paraffin wax for histology.
The removed temporal bone was fixed in 4% paraformaldehyde for 16 h and then decalcified with 10% EDTA in PBS for 2 weeks, after which it was dehydrated and embedded in paraffin wax.
Bones were fixed, decalcified in 14% EDTA, embedded in paraffin, and sectioned and stained with TRAP, toluidine blue, and hematoxylin and eosin.
For histological analyses, undecalcified bones were fixed, deshydrated in ethanol solutions at 4°C and embedded in methyl-methacrylate according to standard protocols.
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