Reprinted from the Archives of Pathology
June 1961, Vol. 71, pp. 611-6114
Copyright 1961, by American Medical Association

Simplified Procedure

for Staining Reticulum

TAMIR K. NASSAR
AND
WILLIAM M. SHANKLIN, Ph.D.
BEIRUT, LEBANON

Fo11owing Mall's differentiation between collagenons and reticular fibers in 1896, many investigators have used silver impregnation to stain reticular fibers. Biondi2 passed frozensections of the brain directly into a solution of potassium permanganate before applying gold chloride sublimate. Perdrau3 treated connective tissue fibers with a 0.25% solution of potassium permanganate before staining with Da Fano's modification of the Bielschowsky method. Foot 4,5 and Wilder6 used potassium permanganate on reticular fibers before applying the del Rio-Hortea silver carbonate method. Using the del Rio Hortega method reticular fibers were stained in the thymus (their Fig. II) by Douthat and Pardinas,7 in the palatine tonsils his Figs. 11-13) by Polak, and in the adrenal cortex (his Figs. 27-28) by del Rio-Hortega.9

In recent studies (Nassar, Issidorides, and Shanklin)10 found that oxidation with equal parts of potassium permanganate and sulfuric acid intensified the silver impregnation of granules in the human neuron. We also found that silver impregnation following oxidation has a high selectivity for reticular fibers. It proved to be especially valuable for staining the reticulum of pathological tissues.

Fig. 1. - Photomicrograph of reticulum of a malignant lymphoma (reticulum-cell type) of the stomach in female, age 38. Formalin fixation; X 250.

Procedure

  1. Formalin-fixed material is embedded in paraffin, sectioned, mounted on slides by egg albumin, dcparaffinized, hydrated, and passed into distilled water.
  2. Oxidize in equal parts of 0.5% potassium permanganate and 0.5% sulfuric acid 1-2 min. until sections turn a brownish color. The solution should be changed frequently.
  3. Rinse in distilled water and decolorize in 2% oxalic acid for 2 min. Wash under tap water 5 min. and pass sections through graded alcohols up to 95%.
  4. Place in 2% silver nitrate, to which pyridine 3 drops / 10 ml have been added, for 0.5 to 1.0 hr. at 50 C. (Good results are also obtained at room temperature at longer periods of time).
  5. Fig. 2. - Photomcrograph of reticulum of the reticular layer of the normal adrenal ; male aged 27. Formalin fixation; X 250.

  6. Rinse quickly in 95% alcohol and impregnate in a silver diammine-hydroxik solution containing pyridine, 3 drops/l0 ml for 5 min. at 50 C.

    Silver Diammine-Hvdroxide Solution (Lillie)11: Place 1 ml. 28% ammonium water in a flask and add 7 ml. 10% solution silver nitrate rapidly. Continue to add 10% silver nitrate drop by drop, shaking between each addition, until a faint permanent turbidity remains after the last drop added. Dilute with an equal volume of distilled water.

  7. Fig. 3. - Photomicrograph of reticulum of normal liver; male, age 20. Forrnalin fixation; X 250.

  8. Rinse quickly in 95% alcohol, and reluce in an equal parts mixture of 2% neutral fornialin and absolute alcohol for 2 mm.
  9. Wash in distilled water and tone in 0.2% aqueous gold chloride until the sections turn grayish.
  10. Fix in 5% sodium thiosuliate for 2 min.
  11. Wash in tapwater and counterstain in Harris' hematoxylin for 5 min.
  12. Decolorize in acid alcohol and wash under tap water until sections turn a blue color.
  13. Dehydrate clear, and mount.

Comment

We have applied this method to the reticulum of many normal and pathological organs. It is relatively simple, and it yields uniformly good results. When organs are impregnated with silver without previous oxidation only a smalll part of the reticulum is impregnated, in addition to nuclei and various inclusions; however, oxidation increases significantly the amount of reticulum revealed and eliminates the impregnation of other tissues. Foot5 found that placing the sections in 0.25% potassium permanganate for 5 minutes, followed by oxalic acid for 10 minutes, sharply intensified the staining of reticular fiber by the silver carbonate impregnation method.

The shorter period of 0.5 hr. in 2% silver nitrate facilitated the staining of the finer fibrils, but after the longer period of 1 hr, relatively fewer fine fibrils were seen; however, there was an increase in the number of coarse fibers.

Members of our Department of Pathology confirm our findings that this method is more satisfactory for staining the reticulum than the current methods used by them.

Lascano12 recently published a silver impregnation method for use after oxidation to stain connective tissue fibers.

Summary

Paraffin sections mounted on slides and oxidized with potassium permanganate and sulfuric acid were placed in 2% silver nitrate, then impregnated with silver diammine. This method demonstrated selectively and exclusively the reticulum of both normal and pathological tissues with great clarity.

The writers wish to thank Dr. Philip Sahyoun, Professor of Pathology, and Dr. Henry Azar, Assistant Professor of Pathology, for their encouragement and for supplying us with the material used in this study.

William M. Shanklin, Ph.D., Department of Histology. School of Medicine, American Univsity of Beirut, Beirut, Lebanon.

REFERENCES

  1. Mall, F. P.: Reticulated Tissue, and Its Relation to the Connective Tissue Fibers, Johns Hopkins Hosp. Rep. 1:171-208, 1896.
  2. Biondi, G.: Un nuovo metodo d'impregnazione del tessuto connettivo in special modo del sistemo nervoso, Riv. Ital. Neuropat. 10:1-9, 1917.
  3. Perdrau, J. R.: The Silver Reduction Method for the Demonstration of Connective Tissue Fibres, J. Path. Bact. 24; 117, 1921.
  4. Foot, N.C.: A Technic for Demonstrating Reticulum Fibers in Zenker-Fixed Paraffin Sections, J. Lab, Clin. Med. 9:777-781, 1924.
  5. Foot N. C.: Chemical Contrasts Between Collagenous and Reticular Connective Tissue, Amer. J. Path. 4:525-344, 1928.
  6. Wilder, H. C.: An Improved Technique for Silver Impregnation of Reticulum Fibers, Amer. J. Path. 11:817-819, 1935.
  7. Douthat A, and Pardińas, R: Observaciones acerca de la naturaleza y extension de los reticulos del timo, Arch. Histol. (B. Air.) 1:415-439, 1942.
  8. Polak, M.: Contribucion al estudio histologico de las amigdalas faringea y palatina, Arch. Histol. (B. Air.) 2:171-201, 1943.
  9. del Rio-Hortega, P.: El metodo del carbonato argentico; revision general de sus tecnicas y aplicaciones en histologia normal y patologia: IV. Coloracion histologica e histopatologica general, Arch. Histol. (B. Air.) 1:329-361. 1942
  10. Nassar, T. K.; Issidorides,, M., and Shanklin, W. M.: Concentric Layers in the Granules of Human Nervous Lipofuscin Demonstrated by Silver Impregnation. Stain Techn. 33:15-18, 1960.
  11. Lillie, R. D: A Simplified Method of Preparation of Diammine-Silver Hydroxide for Reticulum Impregnation: Comments on the Nature of the So-Called Sanstization Before Impregnation, Stain Techn. 21, 69-72, 1946.
  12. Lascano, E. F.: Controlled Differentiation of Cells and Connective Tissue Fibers in Silver Staining, Stain Techn. 35:23-29, 1960.

Submitted for publication April 8, 1960

From the Deprtment of Histology, School of Medicine, American University of Beirut.

This investigation was supported by research grant [?] from the National Institute of Neurological Disease and Blindness of the National Institutes of Health, United States Public [?]



The above article is transcribed from a copy at the AUB's Saab Medical Library.


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