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The new bone tissue was stained red using HE staining.

The new bone tissue was stained red using hematoxylin and eosin (H&E) staining and appeared blue with Masson's Trichrome staining, but upon maturation, it would appear red.

Sections (8 μm for bone, 5 μm for all other tissues) were stained with H&E, Alcian blue with an eosin-orange G counterstain, or for TRAP using a TRAP staining kit (Sigma).

In this study, samples from brain, skin, bone and heart tissue were stained with hematoxylin and eosin (H&E) for histopathological analysis (Fig.  1).

Only stromal tissue is stained.

As indicated by X-ray imaging, cranial digital images and histological (HE and Masson) staining analysis, new bone tissues were formed in the defected area after 12 weeks repair.

For the histological staining of picrosirius red, femoral bone tissues were then embedded in paraffin and cut into 7  μm sections in thickness.

For the histological staining of H&E and TRAP, femoral bone tissues were then embedded in paraffin and cut into 5  μm sections in thickness.

About 40 mg new bone tissues were collected from different parts of the tissue engineering bone graft before the histological staining.

New bone tissues were observed after two months, followed by gradient new bone formation over months according to H&E and Masson staining analysis.

In tetrapods, primary bone tissues are formed by lamellar bone, parallel-fibered bone, or woven bone.