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See also: Tissue Stains: 49,
51, 55,
56, 69,
72, 81.
PRINCIPLE OF THE TEST.
To stain paraffinized tissue sections for carbohydrates and mucoprotein,
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.
Running tap water.
Distilled water.
Periodic acid.
Light green, SF yellowish.
Glacial acetic acid.
Hematoxylin crystals.
Alcohol, 100%
Ammonium or potassium alum.
Mercuric oxide (red).
Coleman's Feulgen solution.
Schiff reagent solution.
Harris' hematoxylin.
1% HCl acid alcohol.
Ammonia water.
95% alcohol.
Absolute alcohol.
Xylene or xylene substitute.
Permount or Histoclad.
STEP-BY-STEP DESCRIPTION.
COLEMAN'S FEULGEN SOLUTION
Dissolve 1.0 gm basic fuchsin in 200.0 ml hot distilled
water. Bring to boiling point. Cool and add 2.0 gm potassium
metabisulfite. 10.0 ml normal hydrochloric acid. Let bleach for
24 hours, then add 0.5 gm activated carbon (Norit). Shake for 1
minute and filter through coarse filter paper. Repeat filtration
until solution is colorless. Store in refrigerator.
SCHIFF REAGENT SOLUTION
Dissolve 1.0 gm basic fuchsin in 200.0 ml hot distilled
water. Bring to boiling point. Cool to 50ø C. Filter and add
20.0 ml normal hydrochloric acid. Cool further and add 1.0 gm
anhydrous sodium bisulfite, or sodium metabisulfite. Keep in the
dark for 48 hours until solution becomes straw colored. Store in
refigerator.
TEST FOR SCHIFF REAGENT SOLUTION
Pour a few drops of Schiff reagent solution into 10 ml of
37-40% formaldehyde in a watch glass. If the solution turns
reddish purple rapidly, it is good. If the reaction is delayed
and the resulting color deep blue-purple, the solution is
breaking down.
0.5% PERIODIC ACID SOLUTION
Periodic acid ........................................ 0.5 gm
Distilled water .................................... 100.0 ml
0.2% LIGHT GREEN SOLUTION (STOCK)
Light green, SF yellowish ............................ 0.2 gm
Distilled water .................................... 100.0 ml
Glacial acetic acid .................................. 0.2 ml
LIGHT GREEN SOLUTION (WORKING)
Light Green (stock) ................................. 10.0 ml
Distilled water ..................................... 50.0 ml
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
STAINING PROCEDURE: For digestion procedure see page 171.
1. Deparaffinize and hydrate to distilled water.
2. Oxidize in periodic acid solution for 5 minutes.
3. Rinse in distilled water.
4. Coleman's Feulgen or Schiff reagent solution for 15
minutes.
5. Wash in running water for 10 minutes for pink color to
develop.
6. Harris' hematoxylin for 6 minutes, or light green
counterstain for a few seconds.
Light green is recommended for counterstaining sections in which
fungi are to be stained. Omit step 7 through 11 if light green
is used.
7. Wash in running water.
8. Differentiate in 1% (HCL) acid alcohol; three to ten
quick dips.
9. Wash in running water.
10. Dip in ammonia water to blue sections.
11. Wash in running water for 10 minutes.
12. Dehydrate in 95% alcohol, absolute alcohol,and clear in
xylene, two changes each.
13. Mount with Permount or Histoclad.
RESULTS.
Glycogen, mucin, reticulin, fibrin, or thrombi, colloid droplets,
hyalin of arteriosclerosis, hyalin deposits in glomeruli,
granular cells in the renal arterioles where preserved,
most basement membranes, colloid of pituitary stalks and thyroid,
amyloid infiltration may show a positive reaction: rose to purplish red
Nuclei -blue
Fungi -red
background -pale green (with light green counterstaining).
Remarks: For excellent additional information on colloidal
iron. Alcian blue 8GX and their combination with periodic acid
Schiff reaction see: "The special value of methods that color
both acidic and vicinal hydroxyl groups in the histochemical
study of mucins with revised directions for the colloidal iron
stain, the use of alcian blue 8GX and their combinations with the
periodic acid-Schiff reaction [ref. 35].
Note: A solution of 5% Aqueous Clorox bleach will reduce overstaining
by leucofuchsin. Running tap water will decolorize the light green.
REFERENCES.
1. McManus SP, Masterson J.
Letter: Cellular metachromasia with toluidine blue O
in cultured white cells of cystic fibrosis heterozygotes.
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Ruiz-Moreno JA.
Adequate staining of Trichomonas vaginalis
by McManus' periodic acid-schiff stain.
Am J Clin Pathol. 1973 May;59(5):741-746.
PMID: 4122087.
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Glomerular Localization of Intravenous Alcian Blue.
Anat Rec. 1964 Aug;149:605-608.
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Extraction Resistance of Sudan Stained mast cells
after Previous Acid Treatment.
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6. McManus JF.
The effect of various solvents and other conditions
on periodate oxidation of carbohydrates in the PAS reaction.
Ann Histochim. 1962 Jan-Mar;7:57-59.
PMID: 13932189.
PubMed Entry
7. McManus JF, Mowry RW.
Effects of fixation on carbohydrate histochemistry.
J Histochem Cytochem. 1958 Sep;6(5):309-316.
PMID: 13587972.
PubMed Entry
8. Beckett EB, Bourne GH.
Some observations on normal and pathological human muscle,
using a combined McManus Periodic acid-Schiff reaction
and Sudan Black stain on gelatine sections.
Acta Anat (Basel). 1958;34(1-2):111-124.
PMID: 13594066.
PubMed Entry
9. Cawley EP, Lupton CH jr, Wheeler CE, McManus JF.
Examination of normal and myxedematous skin; use of Mowry's alcian blue-periodic acid-Schiff technique.
AMA Arch Derm. 1957 Nov;76(5):537-543; discussion 543-544.
PMID: 13468783.
PubMed Entry
10. Cawley EP, McManus JF, Lupton CH jr, Wheeler CE.
An examination of skin from patients with collagen disease utilizing the combined Alcian blue-periodic acid Schiff stain.
Invest Dermatol. 1956 Dec;27(6):389-392; discussion, 393-394.
PMID: 13406286.
PubMed Entry
11. McManus JF.
Factors favouring restriction to 1, 2 glycols of materials coloured
by the periodic acid-Schiff reaction.
Nature. 1956 Oct 27;178(4539):914-915.
PMID: 13369572.
PubMed Entry
12. Cawley EP, Wheeler CE, McManus JF, French AJ.
Use of Ritter and Oleson staining method for demonstration
of fungi in paraffin sections.
AMA Arch Pathol. 1954 Jul;58(1):94-97.
PMID: 13170906.
PubMed Entry
13. Seabury JH, Peabody JW jr, Liberman MJ.
The usefulness of the Hotchkiss-McManus stain for the diagnosis
of the deep mycoses.
Dis Chest. 1954 Jan;25(1):54-69.
PMID: 13116855.
PubMed Entry
14. Muskatblit E, Taschdjian CL, Franks AG.
Hotchkiss-McManus stain versus clearing in KOH in the diagnosis
of superficial mycoses.
AMA Arch Derm Syphilol. 1953 May;67(5):507-509.
PMID: 13050158.
PubMed Entry
15. McManus JF, Mowry RW.
Sulfuric acid hematoxylin stain for basement membranes.
Lab Invest. 1952;1(2):208-209.
PMID: 14939738.
PubMed Entry
16. McManus JF, Lupton CH jr, Graham LS jr.
The demonstration of the intercapillary space
of the human renal glomerulus.
Anat Rec. 1951 May;110(1):57-63.
PMID: 14838331.
PubMed Entry
17. Kligman AM, Mescon H, Delamater ED.
The Hotchkiss-McManus stain for the histopathologic diagnosis
of fungus diseases.
Am J Clin Pathol. 1951 Jan;21(1):86-91.
PMID: 14799469.
PubMed Entry
18. McManus JF, Cason JE.
Carbohydrate histochemistry studied by acetylation techniques.
J Exp Med. 1950 Jun 1;91(6):651-654.
PMID: 15422090.
PubMed Entry
19. McManus JF, Saunders JC, et al.
A hitherto undescribed coloring reaction of certain human nerve fibers.
Science. 1950 Feb 10;111(2876):155.
PMID: 15409108.
PubMed Entry
20. McManus JFA.
Stain Technology 1948;23:99-108.
(AFIP modification), Williams and Wilkins Co.
21. Mowry RW, Kent SP.
Aldehyde pararosanilin (true aldehyde fuchsin) stains purified insulin,
oxidized or not, following adequate fixation with either formalin
or Bouin's fluid.
Stain Technol. 1988 Sep;63(5):311-323.
PMID: 2464218.
PubMed Entry
22. Mowry RW.
Selective staining of pancreatic beta-cell granules.
Evolution and present status.
Arch Pathol Lab Med. 1983 Sep;107(9):464-468.
PMID: 6192790.
PubMed Entry
23. Mowry RW, Longley JB, Emmel VM.
Only aldehyde fuchsin made from pararosanilin stains pancreatic B cell
granules and elastic fibers in unoxidized microsections: problems caused
by mislabelling of certain basic fuchsins.
Stain Technol. 1980 Mar;55(2):91-103.
PMID: 6157231.
PubMed Entry
24. Mowry RW.
Report from the president. The biological stain commission:
its goals, its past and its present status.
Stain Technol. 1980 Jan;55(1):1-7.
PMID: 6158140.
PubMed Entry
25. Alexander WJ, Mowry RW, Cobbs CG, Dismukes WE.
Prosthetic valve endocarditis caused by Histoplasma capsulatum.
JAMA. 1979 Sep 28;242(13):1399-1400.
PMID: 480564.
PubMed Entry
26. Shin MS, Mowry RW, Bodie FL.
Osteosclerosis (punctate form) in multiple myeloma.
South Med J. 1979 Feb;72(2):226-228.
PMID: 424801.
PubMed Entry
27. Mowry RW.
Aldehyde fuchsin staining, direct or after oxidation: problems
and remedies, with special reference to human pancreatic B cells,
pituitaries, and elastic fibers.
Stain Technol. 1978 May;53(3):141-154. Review.
PMID: 83035.
PubMed Entry
28. Wilkoff LJ, Peckham JC, Dulmadge EA, Mowry RW, Chopra DP.
Evaluation of vitamin A analogs in modulating epithelial differentiation
of 13-day chick embryo metatarsal skin explants.
Cancer Res. 1976 Mar;36(3):964-72.
PMID: 1253183.
PubMed Entry
29. James TN, Beeson CW 2nd, Sherman EB, Mowry RW.
Clinical conference: De subitaneis mortibus.
XIII. Multifocal Purkinije cell tumors of the heart.
Circulation. 1975 Aug;52(2):333-344.
PMID: 168001.
PubMed Entry
30. Mowry RW, Kasten FH.
Conference report: The importance of dye purification
and standardization in biomedicine.
Stain Technol. 1975 Mar;50(2):65-81.
PMID: 49946.
PubMed Entry
31. Scott JE, Mowry RW.
Alcian blue--a consumers' guide.
J Histochem Cytochem. 1970 Nov;18(11):842.
PMID: 4100993
PubMed Entry
32. Constantine VS, Mowry RW.
Selective staining of human dermal collagen.
II. The use of picrosirius red F3BA with polarization microscopy.
J Invest Dermatol. 1968 May;50(5):419-423.
PMID: 4172462.
PubMed Entry
33. Constantine VS, Mowry RW.
Selective staining of human dermal collagen.
I. An analysis of standard methods.
J Invest Dermatol. 1968 May;50(5):414-418.
PMID: 4172461.
PubMed Entry
34. Mowry RW, Scott JE.
Observations on the basophilia of amyloids.
Histochemie. 1967;10(1):8-32.
PMID: 4174360.
PubMed Entry
35. Mowry RW.
Ann NY Acad Sci. 1963;106:402-423.
36. Cawley EP, Mowry RW, Lupton CH jr, Wheeler CE.
The remarkable tissue mast cells; with observations on mast cell
acid polysaccharides in the cutaneous lesions of urticaria pigmentosa.
Arch Dermatol. 1959 Dec;80:725-730.
PMID: 13808638.
PubMed Entry
37. Mowry RW.
Improved procedure for the staining of acidic polysaccharides
by Muller's colloidal (hydrous) ferric oxide and its combination
with the Feulgen and the periodic acid-Schiff reactions.
Lab Invest. 1958 Nov-Dec;7(6):566-576.
PMID: 13599446.
PubMed Entry
38. McManus JF, Mowry RW.
Effects of fixation on carbohydrate histochemistry.
J Histochem Cytochem. 1958 Sep;6(5):309-316.
PMID: 13587972.
PubMed Entry
39. Mowry RW.
Observations on the use of sulfuric acid in ether for the sulfation of hydroxyl groups in tissue sections.
J Histochem Cytochem. 1958 Mar;6(2):82-83.
PMID: 13514050.
PubMed Entry
40. Beaird J, Mowry RW, Cunningham JA.
Congenital rhabdomyoma of the heart; case report
with histochemical study of tumor polysaccharide.
Cancer. 1955 Sep-Oct;8(5):916-920.
PMID: 13261044.
PubMed Entry
41. Mowry RW, Millican RC.
A histochemical study of the distribution and fate of dextran
in tissues of the mouse.
Am J Pathol. 1953 May-Jun;29(3):523-545.
PMID: 13040491
PubMed Entry
42. Mowry RW.
Histochemical methods for water-soluble, alcohol-insoluble polysaccharides and other substances oxidized to aldehydes by periodic acid.
J Natl Cancer Inst. 1952 Aug;13(1):230-231.
PMID: 14946518.
PubMed Entry
43. Millican RC, Stohlman EF, Mowry RW.
A comparison of plasma substitutes (dextran, polyvinylpyrrolidone
and oxypolygelatin) with saline therapy in treatment of experimental
tourniquet and burn shock in mice.
Am J Physiol. 1952 Jul;170(1):173-178.
PMID: 12985881.
PubMed Entry
44. Mowry RW, Longly JB, Millican RC.
Histochemical demonstration of intravenously injected dextran
in kidney and liver of the mouse.
J Lab Clin Med. 1952 Feb;39(2):211-217.
PMID: 14898147.
PubMed Entry
45. McManus JF, Mowry RW.
Sulfuric acid hematoxylin stain for basement membranes.
Lab Invest. 1952;1(2):208-209.
PMID: 14939738.
PubMed Entry
46. Mowry RW, Bangle R Jr.
Histochemically demonstrable glycogen in the human heart,
with special reference to glycogen storage disease and diabetes mellitus.
Am J Pathol. 1951 Jul-Aug;27(4):611-625.
PMID: 14846914.
PubMed Entry
47. Morgan C, Mowry RW.
Demonstration of glycogen in the human liver by the electron microscope.
Proc Soc Exp Biol Med. 1951 Apr;76(4):850-852.
PMID: 14844369.
PubMed Entry
48. Totty BA.
Mucins. Chapter 10, pp. 163-200.
In: Bancroft JD, Gamble M.
Theory and Practice of Histological Techniques. Fifth Edition.
Edinburgh: Churchill Livingstone. 2002;:163-200.
ISBN 0-443-06435-0, 796 pages.
49. Best F.
Über Karminfärbung des Glykogens und der Kerne.
[German: Regarding carmine-staining of glycogen and nuclei].
Z f wissenschaftl Mikroskopie u mikroskop Technik. 1906;23:319-322.
50. Casella C.
Il permanganato di potassio quale ossidante in alcune reazioni
istochemiche dei pollisaccaridi. [Italian: Potassium permanganate
as oxidant in some histochemical reactions of polysaccharides].
Anatom Anz. 1942;93;289-299.
51. Cook HC.
A comparative evaluation of the histological demonstration of mucin.
J Med Lab Technol. 1959;16:1-6.
52. Cook HC.
Demonstration techniques in cellular pathology:putting things
into perspective.
Med Lab Sci. 1990;47:210-219.
53. Gomori G.
Microchemical demonstration of iron.
Am J Physiol. 1936;13:665.
54. Gomori G.
Silver impregnation of reticulum in paraffin sections.
Am J Physiol. 1937;13:993.
55. Gomori G.
A new histochemical test for glycogen and mucin.
Am J Clin Pathol. 1946;16:177.
56. Gomori G.
Aldehyde-fuchsin: a new stain for elastic tissue.
Am J Clin Pathol. 1950;20:665.
57. Gomori G.
Microscopic Histochemistry.
Chicago: Chicago University Press. 1952;:.
58. Grocott RG.
A stain for fungi in tissue sections.
Am J Clin Pathol. 1955;25:975.
59. Hale CW.
Histochemical demonstration of acid mucopolysaccharides in animal tissue.
Nature (Lond). 1946;157:802.
60. Horobin RW.
Understanding Histochemistry: Selection, Evaluation and Design
of Biological Stains.
Chchester, UK: Ellis Horwood. 1988;:.
61. Langhans C.
Über Glykogen in pathologischen Neubildungen und den menschlichen
Eihauten. [German: Regarding glycogen in pathologic neoplasms
and in human amnions].
Virchows Arch f patholog Anat u Physiol. 1890;120:28.
62. Mowry RW.
Observations on the use of sulphuric ether for the sulphation
of hydroxyl groups in tissue sections.
J Histochem Cytochem. 1956;4:407.
63. Mowry RW.
Alcian blue techniques for the histochemical study
of acidic carbohydrates.
J Histochem Cytochem. 1958;6:82.
64. McManus JFA, Mowry RW
Staining Methods, Histologic and Histochemical.
London: Harper & Row. 1964;:268.
65. Mowry RW
Aldehyde fuchsin staining, direct or after oxidation: problems
and remedies with special reference to human pancreatic B cells, pituitaries,
and elastic fibers.
Stain Technology. 1978;53:141-154.
66. 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.
67. 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.