Oil red O is a lipid soluble dye, showing much more soluble in the lipid than in the vehicular solvent. Based on this physical property, triglyceride in cells of tissues are specifically stained and differentiated by oil red O, presented as red.
Oil Red O solution
1 Fix frozen sections
Fresh frozen sections are dried to slides in air. Fix slides in formalin for 15 min, briefly wash with running water for 1-5 min, air dry.
2 Oil Red O stain
Put slides in Oil Red O staining solution to stain for 8-10 min under dark condition, wash with distilled water.
Differentiate in 75% alcohol for a few seconds, wash with distilled water.
4 Hematoxylin stain
Counterstain with hematoxylin for 3-8 min, wash with distilled water, and then quickly differentiate in 1% hydrochloric acid alcohol acid for 1 second. Wash in distilled water, bluing with 0.2% ammonia water and wash.
Mount in glycerine jelly.
6 Microscopy examination and analysis
Lipid shows orange to sharp red and blue for nucleus.
Hepatic tissue stained with Oil Red O. Lipid id red and nucleus is blue.
1 For freshly tissue frozen section, firstly fix with formalin followed by stain. If tissue is firstly fixed with formalin before frozen section, air dry sections and then stain.
2 Be gently during the whole process. Rude action may result in lipid loss or shift.
3 The obtained staining slides cannot be preserved long-term. Carry out the microscopy examination as soon as possible after mount.
4 The glycerine jelly is solid under room temperature, and it is better preserved at 60℃ in oven. If bubbles produce during mounting, don’t press or pull apart the coverslip to avoid lipid shift. Immerse the slides in warm water to let the coverslip break off. Then remount the slides with glycerine jelly.
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