Alcian blue

Cells from groups A, B, C, and D were cultured in 6-well plates for 72 hours. Morphological changes in the cells were then observed under an inverted microscope (Olympus, Japan).
Proteoglycans were evaluated with Alcian blue staining. Briefly, each well was hydrated through a graded ethanol series and stained with 1% (w/v) Alcian blue (Sigma, USA) solution in 3% (w/v) glacial acetic acid, pH 4.2, for 30 minutes, followed by washing with tap water for 10 minutes. The results were analyzed using Image J software to semi-quantify the average light density [integral OD (IOD)/area] values.

To detect extracellular polysaccharides, a 100-μL aliquot of the cell culture, harvested from the main cultivation, was suspended in 20 μL India ink (Daiso Co., Ltd., Japan) and observed under an optical microscope (BX53: Olympus, Tokyo, Japan) at DIC (differential interference contrast) conditions with a shutter speed of 0.48 s. Alcian blue (#015-13805: Wako Co., Ltd., Osaka, Japan) staining was conducted for detecting acidic polysaccharides [[21] , [22] (link), [23] (link)]. PAS (#164-19705: Wako Co., Ltd.) staining was performed for detecting neutral polysaccharides [24 ]. Cells stained with Alcian blue or PAS were observed using BX53 (Olympus) at DIC conditions with a shutter speed of 0.48 s. Regarding intracellular lipid staining [25 (link)], a 1-mL aliquot of the cell culture was collected from the main cultivation and mixed with 20 μM of Nile red (#144-08811: Wako Co., Ltd.). Samples were observed for morphology using an optical photomicroscope at a high resolution with a Nomarski prism or using the fluorescence microscope, BX53/DP72, fitted with a special filter, U-FBW (Olympus) for excitation (460–495 nm) and emission (510 nm). The exposure time for photography was 0.2 s.