[實驗技術] 油紅實驗 （Oil red O staining）

Oil red O staining （12 well -scale, Collagen coated plate）

Preparation

1.   99% isopropanol and 60% isopropanol

2.   Sterilised PBS buffer 500 mL

3.  4% paraformaldehyde (PFA) in PBS buffer, pH 7.4 (FRESH MADE)

4.   Oil red O solution

A.       (Stock): 300 mg oil red O powder in 100 mL 99% isopropanol 

        ( stable for one year ) >>>3 mg/mL

B.       (Working): 30 mL stock Oil red o solution: 20 mL deionized water (DW)

(stable for only for two hours, FRESH MADE) >>> Final conc.1.8 mg/mL 

Process

1.    PBS 1 mL/ well wash x 2, washing gently and voiding the cell floating.

2.    Aspirate the PBS

3.    Add 4% PFA 1 mL/ well, incubation at room temperature for 1 hour

4.    Transfer PFA to PFA waste bottle

5.    Add PBS 1 mL/well x 1

6.    Add 60% isopropanol 1 mL/well for 10 mins

7.    Aspirate 60% isopropanol

8.    Add Oil red O working solution 1 mL/ well, and incubation for 10 min (If it is possible, put the plate on the Microplate shaker, slowly)

9.    Aspirate Oil red O working solution, and wash with PBS x1 or until the waste is clear

10. Elute Oil red O by adding 100 % isopropanol 1 mL/well

11. Incubation for 30 min at room temperature

12.Transfer 200 uL of each well to 96 well plate for analysis

13. Measure OD at 500 nm ( Blank: 100 % isopropanol)