Neutral formalin solution

To calculate metastatic nodules, tumors in mice were collected and washed with PBS, followed by fixed in 10% neutral formalin solution (Sigma-Aldrich) and embedded in paraffin (Sigma-Aldrich). Tissues were sliced, after which were stained by Hematoxylin-Eosin (HE) (Sigma-Aldrich).

The left lobe of the liver was fixed in 10% neutral formalin solution (Sigma-Aldrich) overnight at 4 °C, cryopreserved in a 30% (w/v) sucrose solution for 24 h at 4 °C, and stored at −80 °C. Seven μm-thick liver sections were cut with a refrigerated microtome (Leica), collected on poly-L-lysine–coated glass slides, and stored at −80 °C until staining. Oil Red O lipid stain was done with a 5% solution of Oil Red in Propylene glycol for 10 min, in a heater, at 60 °C, followed by a 5 min of wash in 85% Propylene glycol solution (Oil Red O and Propylene glycol - Sigma). Hematoxylin–eosin (H&E) counterstaining was done on frozen slides with Mayer hematoxylin (Bio-Optica) for 1 min and, after water rinsing, with 1% eosin aqueous solution (Bio-Optica) for 4 min. After staining, the slides were cleared in xylenes and cover slipped with xylenes-based mounting medium (Eukitt, Bio-Optica). The liver sections were evaluated in blind by light microscopy. Images of the stained sections were captured using Microscope Axioscop2 mot plus (Zeiss). Quantitative analysis of lipid droplet-stored triglycerides was done using ImageJ imaging software^{55 (link)}.

After sacrifice, the specimen containing the bone graft was fixed with neutral formalin solution (Sigma Aldrich, St. Louis, MO, USA) for two weeks and then removed calcium using the EDTA solution (10%, pH 7.0) after cleaning by distilled water. After calcium removal was confirmed, the ethanol concentration was increased for dehydration. Then, dealcoholization, paraffin infiltration, and paraffin embedding were performed in order. The paraffinized specimen block was sectioned longitudinally in the center of each defect using a microtome, and then mounted on the slide. The thickness of the slides produced was 4 μm. Hematoxylin-eosin (H-E) staining and Masson’s trichrome staining were performed to observe the newly regenerated bone tissues. The most central area was selected from each block for histologic and histomorphometric evaluation. The images on selected slides were saved using an optical microscope connected to a computer (BX51, OLYMPUS, Tokyo, Japan) and a CCD camera (SPOT Insight 2Mp scientific digital camera system, DIAGNOSTIC Instruments Inc., Sterling Heights, MI, USA). The saved images were analyzed by i-Solution ver. 8.1 (IMT i-Solution, Inc., Coquitlam, BC, Canada). Typical specimen images were observed at ×12.5 magnification. For histomorphometric analysis, ×40 and ×400 magnifications were used. New bone area percentage (%) in the defect was analyzed and recorded.