油红染色.docVIP

Oil Red O Staining Protocol ? Description: This protocol is for lipid and fat staining on frozen sections. It may not suitable for paraffin embedded tissue sections. Fixation: 10% formalin (fixation is necessary, damage of unfixed sections has been seen) ?Section: Frozen sections at 5-10 um thick ? Solutions and Reagents:? 0.5% Oil Red O Solution: ? ????? Oil Red O -------------------------- 0.5 g ????? Propylene glycol, 100% ----------- 100 ml ????? (Propylene glycol is also called 1,2-Propanediol, Sigma Cat# 398039-500ML or Cat# P4347) ? ????? Add a small amount of propylene glycol to the oil red O and mix well, crush larger pieces with stirring bar. Gradually add the remainder of the propylene glycol while stirring. Heat gently until the solution reaches 95 -100 oC. Do not allow to go over 110 oC. Overheating will result in high background staining. Filter solution through coarse filter paper (i.e. 25 um filter paper) while still warm. Allow to stand overnight at room temperature. The solution can be stored at room temperature for many years. If precipitate forms in the solution, re-filter. ? 85% Propylene Glycol Solution: ? ????? Propylene glycol, 100% ----------------- 85 ml ????? Distilled water -------------------------- 15 ml ? Gills or Mayers Hamatoxylin Solution ? Procedure: Cut fresh frozen tissue sections at 5-10 um thick and mount on slides. Air dry slides for 30-60 minutes at room temperature and then fix in ice cold 10% formalin for 5-10 minutes. Air dry again for another 30-60 minutes or rinse immediately in 3 changes of distilled water. Let slides air dry for a few minutes. Place in absolute propylene glycol for 2-5 minutes to avoid carrying water into Oil Red O. Stain in pre-warmed Oil Red O solution for 8-10 minutes in 60 oC oven. Differentiate in 85% propylene glycol solution for 2-5 minutes. Rinse in 2 changes of distilled water. Stain in Gills or Mayers hamatoxylin for 30 seconds. Wash thoroughly in running tap water for 3 minutes. Place sli