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United States Government Work, uncopyrighted, public-domain,
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PRINCIPLE OF THE TEST.
To stain paraffinized tissue sections
for fat, required in the diagnosis of certain diseases
encountered in anatomic pathology.
SPECIMEN REQUIRED.
All human tissue excised at surgery, outpatient clinics, and postmortems,
fresh or in fixative, along with a filled-out Tissue Examination Form
(U. S. Standard Form 515, USSF515).
REAGENTS, INSTRUMENTATION.
Oil red O.
Propylene glycol.
Hematoxylin crystals.
Sodium iodate.
Ammonium or potassium alum.
Citric acid.
Chloral hydrate.
Alcohol, 100%.
Mercuric oxide (red).
STEP-BY-STEP DESCRIPTION.
FlXATION: 10% buffered neutral formalin.
TECHNIQUE: Frozen sections.
SOLUTIONS: 0.5% OIL RED O SOLUTION
Oil red O........................................... 0.5 gm
Propylene glycol, 100%............................ 100.0 ml
Add a small amount of propylene glycol to the oil red O and mix well, crush
larger pieces. Gradually add the remainder of the propylene glycol, stirring
periodically. Heat gently until the solution reaches 95oC.
Do not allow to go over 100oC. Stir while heating.
Filter through coarse filter papor while still warm.Allow to stand overnight
at room temperature.
Filter through Seitz filter with the aid of vacuum. If solution becomes
turbid, refilter. When using the Seitz filter, put rough surface of
filter up.
85% PROPYLENE GLYCOL SOLUTION
Propylene glycol, 100%............................. 85.0 ml
Distilled water.................................... l5.0 ml
MAYER'S HEMATOXYLIN SOLUTION
Hematoxylin crystals................................ 1.0 gm
Distilled water.................................. 1000.0 ml
Sodium iodate....................................... 0.2 gm
Ammonium or potassium alum......................... 50.0 gm
Citric acid......................................... 1.0 gm
Chloral hydrate.................................... 50.0 gm
HARRIS' HEMATOXYLIN SOLUTION
Hematoxylin crystals................................ 5.0 gm
Alcohol, 100%...................................... 50.0 ml
Ammonium or potassium alum........................ 100.0 gm
Distilled water.................................. 1000.0 ml
Mercuric oxide (red)................................ 2.5 gm
5% ACID WATER SOLUTION
Hydrochloric acid................................... 5.0 ml
Distilled water.................................... 95.0 ml
GLYCERIN JELLY
Gelatin............................................ 10.0 gm
Distilled water.................................... 60.0 ml
Heat until gelatin is dissolved. Add:
Glycerin........................................... 70.0 ml
Phenol.............................................. l.O ml
STAINING PROCEDURE
1. Cut frozen sections and collect in distilled water.
2. Absolute propylene glycol for 2 minutes.
3. Oil red O solution for 1 hour. Note: If sections are mounted on glass
slides before staining, allow to stand in ORO overnight.
4. Differentiate in 85% propylene glycol solution for 1 minute.
5. Rinse in distilled water, two changes.
6. Stain in Mayer's or Harris' hematoxylin solution for few seconds.
7. Rinse in distilled water, two changes.
8. Differentiate in acid water solution, if overstainer.
9. Wash in water.
10. Neutralize in weak ammonia water if differentiated in acid water sol.
11. Wash in water, two changes.
12. Mount with glycerin jelly.
CONTROL: Adipose tissue
RESULTS:
Fat: red.
Nuclei: blue.
REFERENCES.
1. Jones ML.
Lipids. Chapter 11, pp. 201-230.
In: Bancroft JD, Gamble M.
Theory and Practice of Histological Techniques. Fifth Edition.
Edinburgh: Churchill Livingstone. 2002;:201-230.
ISBN 0-443-06435-0, 796 pages.
2. Baker JR.
Structure and chemical composition of the Golgi element.
Q J Microscop Sci. 1946;87:441.
3. Benda C.
Eine makro- und mikroskopische Reaktion der Fettgewebsnekrose.
[German: A macroscopic and microscopic reaction of fat tissue necrosis].
Virchows Arch f patholog Anat u Physiol u f klin Medizin. 1900;:161.
4. Bodian M, Lake BD.
The rectal approach to neuropathology.
Br J Surgery. 1963; 50:702.
5. Cain AJ.
Use of Nile Blue in the examination of lipoids.
Q J Microscop Sci. 1947;88:383.
6. Daddi L.
Nouvelle méthode pour colorer la graisse dan les tissues.
[French: New method for staining fat in tissues].
Arch Ital de Biologie. 1896;26:143.
7. Feulgen R, Voit K.
Über einen weitverbreiteten festen Aldehyd, seine Entstehung
aus einer Vorstufe, seine mikrochemischer Nachweis, und die Wege zu seiner
präperativen Darstellung. [German: Regarding a widely-distributed,
fixed aldehyde, its emergence from precursors, its microchemical
demonstration, and the pathways to its preparational demonstration].
Pflügers Arch f d gesamte Physiol d Menschen u d Tiere.
1924;206:389.
8. Fischler FJ
Über die Unterscheidung von Neutralfetten, Fettsäuren,
and Seifen in Gewebe. [German: Regarding the discrimination of neutral fats,
fatty acids, and soaps in tissue].
Zentralbl f allgem Pathologie u pathol Anat. 1904;15:913.
9. Michaelis L.
Über Fett-Farbstoffe. [German: Regarding fat-stains].
Virchows Arch Pathol Anat u Physiol u f klin Medizin. 1901;164:263.
10. Schultz A.
Eine Methode des mikrochemischen Cholesterin-nachweises an Gewebsschnitt.
[German: A method for microchemical demonstration of cholesterol
in tissue sections].
Zentralbl f allgem Pathologie u pathol Anat. 1924:35:314.
11. Smith JL.
On the simultaneous staining of neutral fat and fatty acid
by oxazine dyes.
J Pathol Bacteriol. 1908;12:1.
12. Verne J.
Étude histochemique des substances aldéhydiques
formées au cours du metabolisme des corps gras.
[French: Histologic investigation of aldehyde substances formed by the
metabolism of fat bodies].
Ann de Physiol. 1929;5:245.
13. Prophet EB, Mills B, Arrington JB, Sobin LH, eds.
Laboratory Methods in Histotechnology.
Washington, DC: Armed Forces Institute of Pathology. 1992;:.
ISBN 1-881041-00-X, 278 pages.
14. Mikel UV, ed.
Advanced Laboratory Methods in Histology and Pathology.
Washington, DC: Armed Forces Institute of Pathology. 1994;:.
ISBN 1-881041-13-1, 254 pages.