After micro-CT scanning, samples were dehydrated and made transparent using dimethylbenzene (Sinopharm Chemical Reagent Co., Ltd.). They were then embedded in wax and sectioned into 6 µm coronal planes. Sections were stained with hematoxylin and eosin (H&E) and observed under a light microscope (Leica Microsystems GmbH, Wetzlar, Germany). Image-Pro Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA) was used to evaluate new bone formation at ×100 magnification in six randomly selected fields per section. Bone density was defined as the ratio of new bone area to total area. The border of the new bone and osteoid tissue was difficult to define, so osteoid tissue was not included in new bone calculations.
Immunohistochemistry was performed using antibodies specific for CD31 (1:200; ab24590; Abcam) and HIF-1α (1:100; ab8366; Abcam). In brief, these sections were rehydrated and incubated with primary antibodies at 4°C overnight, then incubated with biotinylated secondary IgGs (1:500; BA1001; Wuhan Boster Biological Technology, Ltd., Wuhan, China). Sections were treated with ABC complex and developed with 3,3′-diaminobenzidine (both Wuhan Boster Biological Technology, Ltd.), then stained with hematoxylin. All sections were consistently maintained in liquid, and sections incubated without primary antibodies were used as a control.
Immunohistochemistry was performed using antibodies specific for CD31 (1:200; ab24590; Abcam) and HIF-1α (1:100; ab8366; Abcam). In brief, these sections were rehydrated and incubated with primary antibodies at 4°C overnight, then incubated with biotinylated secondary IgGs (1:500; BA1001; Wuhan Boster Biological Technology, Ltd., Wuhan, China). Sections were treated with ABC complex and developed with 3,3′-diaminobenzidine (both Wuhan Boster Biological Technology, Ltd.), then stained with hematoxylin. All sections were consistently maintained in liquid, and sections incubated without primary antibodies were used as a control.