4.4. Morphology:Lipid (Oil Red O) Staining

A lipid staining kit was used for selective staining and detection of lipid droplets in matured 3T3-L1 cells (matured adipocytes). Oil red O is a lysochromediazo dye used for staining lipids. Hematoxylinwas included in the kit stains the nuclei of the cells. The Lipid (Oil Red O) Staining Kit contained PBS, 10% formalin, Oil Red O and hematoxylin (Sigma Alrich). Other reagents and supplies required were Whatmann No. 1 filter paper, 60% isopropanol and 100% isopropanol. The experimental protocol is briefly described below:

On Day 28 of cell culture, immediately after the media collection from all of the wells, cells were ready for staining. The staining assay started with the remaining cell culture media in the wells being removed. After removal of cell culture media, cells were washed twice with PBS. After the wash, 10% formalin was pipetted down on to the sides of wells. The formalin treated cells were incubated for 45 min. After the incubation period, formalin was discarded and cells were washed twice with water. 60% isopropanol was added to wells and incubated for 5 min. After discarding the 60% isopropanol, cells were evenly covered with Oil Red O working solution (was prepared 15 min before use by adding, mixing and filtering 3 parts of Oil Red O stock solution to 2 parts of water). The well plate was gently rotated and incubated for 15 min. After the incubation period, the Oil Red O solution was discarded and stained cells were washed five times with water until no excess stain was seen. After obtaining clear cells, hematoxylin was added to the cells and incubated for 1 min, after which thehematoxylin was discarded and the cells were washed five times with water. Cells were covered with water and observed under a microscope. Images were captured at the time of observation. Lipid droplets appeared red and nuclei appeared blue.