Histobond

BP explants designated for IHC were fixed in 10% neutral buffered formalin at 4°C for 48 hours. The tissue was then gradually dehydrated and embedded in paraffin. The paraffin blocks were sectioned at 6 μm and mounted on Histobond © (VWR; Radnor, PA) slides. The slides were then heated in an oven and rehydrated in successive xylene to ethanol baths. The slides were incubated overnight in 60°C citrate buffer (Thermo Fisher; Waltham, MA) for antigen retrieval. Following antigen retrieval, slides were washed, blocked with goat serum (Abcam ab7481), rinsed with wash buffer, and incubated in primary antibodies (αAGE, 400 ng/ml| Abcam 23722; αBSA, 38 ng/ml| Abcam 192603; αCD68| Sino Biological 80307-R001) overnight at 4 °C. After incubation with primary antibodies, slides were washed and incubated with 3% hydrogen peroxide for 10 minutes. Slides were then rinsed and incubated for 1 hour at room temperature with the appropriate horseradish peroxidase polymer-conjugated secondary antibody (Abcam ab214880; Cambridge, United Kingdom). Slides were then rinsed and incubated for 8 minutes at room temperature with 3,3’Diaminobenzidine substrate (Abcam ab64238; Cambridge, United Kingdom). Slides were then counter-stained using regressive hematoxylin staining, dehydrated, and cover-slipped. Serial sections were stained with H&E for morphologic assessment.

For visualization of calcium deposits on rat BP explant microscopic sections for both 7-day and 28-day rat explants, Alizarin Red staining solution was used (Sigma A5533). Briefly, BP explants designated for IHC were fixed in 10% neutral buffered formalin at 4°C for 48 hours. The tissue was then gradually dehydrated and embedded in paraffin. The paraffin blocks were sectioned at 6 μm and mounted on Histobond © (VWR; Radnor, PA) slides. The slides were then heated in an oven and rehydrated in successive xylene to ethanol baths. Slides were then rinsed in water for 5 minutes and dipped in a 40 mM Alizarin Red water solution. Slides were quickly rinsed with water for 5 minutes, dehydrated in consecutive ethanol to xylene baths, and cover slipped.