4.1 OIL RED O FOR THE DEMONSTRATION OF LIPIDS
This method is suitable for tissue containing lipids in tiny droplets. Lipids dissolve less rapidly in this staining solution than in the Sudan Technique.

PRINCIPLE
Lipids are soluble in the solvents used in standard histological tissue processing, therefore cryostat sections on fresh or fixed tissue are used. Oil-soluble dyes stain lipids by being more soluble in the lipids than in their solvents.

REFERENCES
Flinders Medical Centre - Modified Oil Red O Technique p 108 Cook H C, Manual of Histological Demonstration Techniques. p 360 Culling CFA, Handbook of Histopathological and Histochemical Techniques, 3rd ed SPECIMEN 1. 6 - 8 æ cryostat sections on fixed tissue. Dry sections onto slides for at least one hour. 2. Cryostat sections on fresh tissue post fixed for one hour in formal calcium.

CONTROL
Liver with fatty changes fixed in 10 % neutral buffered formalin.

REAGENTS
(A) OIL RED O STOCK SOLUTION Oil red O (C.I 26125) 0.5 g Isopropanol 500 mL Warm the dye and alcohol in a long necked flask over a water bath at 56øC for 30 - 60 mins. Cool.
OIL RED O WORKING SOLUTION Stock solution 30 mL OR 12 mL Distilled water 20 mL 8 mL Mix well and stand for 5 to 10 mins. Filter. Use same day.
(B) FORMAL CALCIUM 40 % formaldehyde 100 mL Distilled water 900 mL 10 % Calcium chloride 100 mL
(C) HARRIS OR MAYER'S HAEMATOXYLIN

PROCEDURE
1. Stain in working solution - 5 - 10 mins in a closed container.
2. Wash well in water but gently (sections tend to float).
3. Harris Haematoxylin - 1/2 - 1 min or Mayer's Haematoxylin 3 minutes.
4. Blue in tap water.
5. Rinse in distilled water.
6. Mount in aqueous mounting medium.
NOTE: Use a closed container when staining in working solution to avoid precipitation on the section due to evaporation of the solvent.

RESULTS
Triglycerides (neutral fats) Deep orange red
Nuclei Blue
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4.2 NILE BLUE SULPHATE METHOD FOR ACIDIC AND NEUTRAL LIPIDS

PRINCIPLE
Nile blue sulphate serves as a preliminary test to indicate the distribution of neutral versus acid lipids. Nile blue sulphate comprises two components; a red oxazone which dissolves in neutral lipids and a blue oxazine which is basic and reacts with phospholipids and free fatty acids.

REFERENCES
p 170 & 223 Bancroft JD & Stevens, Theory & Practice of Histological Techniques, 2nd Ed. p 111 Cook H C, Manual of Histological Demonstration Techniques. p 363 Culling CFA, Handbook of Histopathological and Histochemical Techniques, 3rd Ed.

SPECIMEN
1. 6 - 8 æ frozen sections on fixed tissue post fixed for one hour in formal-calcium.
2. 6 - 8 æ Cryostat sections on tissue fixed in 10 % neutral buffered formalin

CONTROL
Phospholipids myelin Neutral fat subcutaneous tissue or fatty liver.

REAGENTS
(A) NILE BLUE SULPHATE SOLUTION
1% aqueous Nile Blue Sulphate solution should be tested for the presence of oxazone (Nile Red) by shaking a little of it with a little amount of xylol. The xylol, after 20-30 seconds, should be a definite red. A poor oxazone content may be improved by the following method. Add 10mLs of 1% Sulphuric Acid to 200mLs of 1% Nile Blue Sulphate (C.I. 51180) in water. Boil under reflux for 4 hours. The solution should be at pH2 so that non-lipid reaction is minimal.
(B) FORMAL CALCIUM
40 % formaldehyde 100 mL Distilled water 900 mL 10 % Calcium chloride 100 mL
(C) 1% METHYL GREEN, ZINC CHLORIDE SALT
(Crystal Violet Free) Aldrich # 32,382 - 9. or 1% CHLOROFORM-WASHED METHYL GREEN (C.I. 42585) 1% Aqueous methyl green which has been washed in a separating funnel with chloroform until all trace of methyl violet is removed and chloroform remains colourless.

PROCEDURE
1. Cut frozen sections and fix for 1 hour in formal calcium.
2. Stain in Nile Blue Sulphate at 60øC for 30 mins.
3. Differentiate in 1% acetic acid for 1 - 2 mins.
4. Wash well and counterstain nuclei with 1% chloroform-washed methyl green 5 mins.
5 Wash well and mount in glycerine jelly.

RESULTS
Unsaturated hydrophobic lipids Pink
Free fatty acids Pink to blue Phospholipids Blue

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