Oil Red O Staining Kit for Cultured Cells 

Lipids refer to neutral fats, lipids, and their derivatives, which share a common physical property: they are insoluble in water but soluble in organic solvents. Common dyes used for lipid staining in histology include Sudan I, Sudan III, Sudan IV, Sudan Black B, and Oil Red O. While traditional methods often use Sudan dyes, Oil Red O provides better visualization, offering clearer and more distinct staining, making it more suitable for fat staining.

Oil Red O (ORO) is a lipid-soluble azo dye that dissolves in and binds to fats (such as triglycerides), forming orange-red lipid droplets. The staining principle is based on the fact that lipid-soluble dyes have a higher solubility in tissue lipids than in the solvent, allowing the dye to transfer from the solution into the lipid droplets in tissues. Oil Red O is primarily used to display fat degeneration and abnormal lipid deposition in tissues and organs. It is particularly useful for the following applications: Fatty degeneration in solid organs such as the liver, heart, and kidneys; Accumulation of neutral fat droplets within cells; Lipid deposition beneath endothelial cells; Lipid identification in fat emboli; Tumor diagnosis and differentiation in adipose tissue.

MCE Oil Red O Staining Kit for Cultured Cells consists of Oil Red O staining solution and hematoxylin. It is used to demonstrate fat degeneration and abnormal lipid deposition in cultured cells, particularly when multiple neutral fat droplets are present within the cells. This kit helps in identifying lipid changes and their nature in cultured cells. It is important to note that samples should not be fixed with ethanol-containing fixatives, as ethanol may interfere with lipid staining. The positive staining result for fat typically appears orange-yellow to red, with the exact color depending on the lipid concentration.

Reagents Preparation

60% Isopropanol, Distilled Water, PBS (Phosphate Buffered Saline), or Saline Solution, etc.

Procedure

1. Cell Fixation: Fix the cultured cell samples using a cell fixative for 15-25 min.

2. Cell Washing: Wash the cells with PBS or saline solution, then allow them to air dry briefly.

3. Isopropanol Treatment: Treat the cells with 60% isopropanol for 20-30 s.

4. Oil Red O Staining

4.1 Preparation of Oil Red O Working Solution: ORO Staining Solution A and ORO Staining Solution B in a 3:2 (v/v) ratio to prepare the working solution.

Note: The Oil Red O working solution is unstable and may precipitate. It is recommended to prepare it fresh before use. After preparation, let it stand for 20-40 min or centrifuge at 3,000 rpm for 10 min and use the supernatant for staining.

4.2 Oil Red O Staining: Apply the Oil Red O working solution to the cells and stain for 10-15 min, ensuring the staining solution covers the sample.

5. Isopropanol Treatment: Rinse the stained cells briefly with 60% isopropanol to remove excess dye, then wash with PBS or saline solution.

6. Mayer Hematoxylin Staining: Counterstain the nuclei with Mayer Hematoxylin Solution for 2-5 min.

Note: Do not over-stain with hematoxylin.

7. (Optional) Washing: Wash the cells with ORO wash buffer for 1 min.

8. Rinsing and Observation: Rinse the cells with distilled water for 8-10 min, allow them to air dry, and then observe under the microscope.

Note: Staining results should not be stored for long periods; observe and capture images as soon as possible.

Interpretation of Staining Results

1. Neutral Fat: Orange-red or Red-orange.

2. Cell Nuclei: Blue.

1. Reagents should be used as soon as possible after opening to avoid potential effects on experimental results.

2. This product is for R&D use only, not for drug, household, or other uses.;

3. For your safety and health, please wear a lab coat and disposable gloves to operate.