Ts100

As for histology evaluation, animals were euthanized and sciatic nerve was dissected after 12 weeks post implantation. Nerve specimens were fixed with 4% paraformaldehyde for 6 h, dehydrated with ethanol and embedded in paraffin. Then, the samples were cut into 5 μm sections, stained with hematoxylin-eosin (HE), and observed by light microscope (TS100, Nikon, Japan). The regeneration of axon and myelin sheath in sciatic nerve were evaluated by toluidine blue staining. After euthanization, the nerve segments were picked out and fixed with 2.5% glutaraldehyde 2 h, then fixed with 1% osmium tetroxide for 2 h, dehydrated by ethanol and embedded in Epon812. Semi-thin sections were obtained using a glass knife, stained with toluidine blue and observed by light microscope (TS100, Nikon, Japan).

The intracellular lipid contents of 3T3-L1 adipocytes were determined by Oil Red O staining [33 (link)]. Briefly, differentiated 3T3-L1 cells were washed twice with PBS and fixed with 10% formaldehyde for 1 h. After another washing with PBS, the fixed cells were stained with 0.5% Oil Red O in 3:2 of Oil Red O/H₂O for 15 min at room temperature and then washed with 60% isopropanol and distilled water. The lipid content was imaged with an inverted light microscope Nikon TS100 (Tokyo, Japan). Finally, 100% isopropanol was used to elute Oil Red O dye and it was quantified at 492 nm absorbance.

Subsequently, turbidity of NaCNs samples prepared with different concentrations and at different mixing duration was measured to analyze the possible effect of concentration and time on the formation of micelles, at 400 nm using a UV-Vis Spectrophotometer. Turbidity measurement is an indirect method to determine the concentration and time-dependent changes in optical density due to micellar growth in a supersaturated solution [31 (link)]. For turbidity analysis, different amounts of sodium caseinate (0.125, 0.25, 0.50, 1, 2, 4, 6, 8 and 10 mg) were added to 1 mL of distilled water in Eppendorf tubes. Nine samples were prepared, as stated above and placed on the nutating mixer. After a 1 h of mixing, the samples absorbance was measured at 400 nm (optimized) [32 (link)]. Similar procedure was followed after 2, 4 and 8 h of incubation. All measurements were performed at 25 °C in triplicates with the results reported as mean (±SD).
To observe the aggregation patterns among micelles prepared with different concentrations, optical images were taken using an optical microscope (Inverted trinocular microscope Nikon TS100, Minato-Ko, Tokyo, Japan) at a scale bar of 100 μm and a magnification of 10×. Following micelles formation, the samples were transferred to a six well plates, and then microscopic images were taken.