THE JOURNAL OF INVESTIOATIVE DERMATOLOGY Vol. 52, No. 4 Copyright 1969 by The Williams & Wilkins Co. Printed in U.S.A.

A COMBINED TISSUE STAIN FOR THE SELECTIVE OF COLLAGEN, ELASTIC FIBERS AND ACIDIC * V. S. CONSTANTINE, MS., MD.

The histopathologic diagnosis of many skin Dyes diseases depends on the Visualization of con- 1. Acid fuchsin (for mixed collagen stain only) nectiVe tissue components in the skin biopsy 2. Basic fuehsin 3. Rematoxylin specimen. Collagen fibers, elastic fibers, and 4. Alcian blue 8GX (George Gurr) acid mucopolysaccharides form the three ma- 5. Sirius red F3BA (purchased from Verona or extracellular components Dyestuffs; P. 0. Box 385; Springfield Road; of the dermis. A number of techniques are Union, New Jersey). available for staining each of these tissue com- When possible, all dyes used in histologic procedures should be certified by the Biological ponents separately. To study their relationship,Stain Commission (2). Alcian blue SGX and sirius however, it is useful to visualize all three inred F3BA are commercial dyes and are not certi- the same histologic section. Although in thefled. past elastic tissue stains have been combined with a collagen stain or a stain for acidic Chemicals 1. 3% acetic acid carbohydrates (1), a technique for staining all 2. 0.25% and concentrated hydrochloric acid three connective tissue components in the same (llCl) histologic section combining highly selective 3. Saturated aqueous picric acid. The solution stains for each component has not been avail- should be prepared at least several hours able. prior to use and must have some undissolved crystals present. The present communication describes a rela- 4. 29% aqueous ferric chloride U.S.P. tively simple and reproducible technique for 5. Paraldehyde combining highly selective stains for collagen, elastic fibers and acidic carbohydrates for theDye Mixtures histologic study of dermal lesions. 1. Alcian Blue (3) Add one crystal of thymol to prevent fungus growth. The solution keeps at room tempera- MATERTALS AND METHODS ture indefinitely. 2. Gomori's Aldehyde Fuchsin (4) Tissues Used Concentrated HC1 2 ml. For the initial portion of the study while the Paraldehyde 2 ml. technique was being developed, normal skin was 0.5% basic fuchsin in 70% 200 ml. obtained from the abdominal incision of 10 pa- tients at autopsy. The tissue was fixed with neu- Let the solution stand at room temperature tral buffered formalin (1) and processed in a for at least 24 hours. When it turns deep routine manner. Sets of 10 slides each were used purple it is ready for use and must be stored for the various staining trials. in the refrigerator. Filter before each use. The evaluation of the technique is also based Staining becomes pale with extensive use or on the staining of a section from each skin lesion storage for some months. submitted to the surgical pathology laboratory 3. Weigert's Hematoxylin (5) for diagnostic and routine purposes over the Stock Solution A period of approximately 18 months. During this Hematoxylin 1 gm. time minor variations of dye concentration and 95% ethanol 100 ml. staining time were tried for optimal results. Stock Solution B Aqueous ferric chloride (29%) 4 ml. Distilled water 95 ml. Supported in part by a Veteran's Administra- Concentrated HC1 1 ml. tion Research Associateship. Working Solution Received September 21, 1968; accepted for pub- Equal parts of A and B lication October 11, 1968. * From the Departments of Dermatology and The stock solutions keep indefinitely but Pathology, University of Alabama Medical Cen- the working solution deteriorates after 10-14 ter, Birmingham, Alabama 35233. days. 353 354 THEJOURNAL OF INVESTIGATIVE DERMATOLOGY

4. Collagen Stain containing sirius red and acid fuchsin is used, A. Picrosjrjus red F3BA (6) all the collagen is completely stained. The Saturated aqueous picric acid 200 ml. Sirius red F3BA 0.2 gm. smaller fibers stain somewhat more with B. Mixed Collagen Slain sirius red and appear brighter red than the Saturated aqueous picric acid 200 ml. larger fibers which stain more with acid fuchsin Acid fuehsin (pure dye) 0.03 gm.' and stain a somewhat deeper red. Sirius red F3BA 0.05 gm. Acidic carbohydrates stain blue. These in- Concentrated HC1 0.5 ml. Both solutions A and B keep at room tem-clude polyanion substances containing car- perature indefinitely or until exhausted byboxyl groups and/or sulfate groups and sialic extensive use. acid-containing substances (so-called epithelial mucins). Aldehyde fuchsin, which is used to Slaining Procedure stain the elastic fibers, also stains some of the 1. Deparaffinize the tissue sections andacidic substances. Since prior Alcian blue stain- hydrate them in running tap water (RTW) for 10 minutes. ing blocks most of the acidic binding sites, alde- 2. Rinse in 3% acetic acid by dipping thehyde fuehsin stains very little in the dermis staining basket into the solution ten times.other than elastic fibers. Some mast cell 3. Stain in Alcian blue for 30 minutes. granules, sialomuein granules in eccrine sweat 4. Rinse in RTW for 3 minutes. 5. Rinse in 70% alcohol by dipping the stain-coils and fungi were found to have been stained ing basket into it ten times. by aldehydc fuchsin more strongly than with 6. Stain in aldehyde fuchsin for 30 minutes.Alcian blue. Elastic fibers and internal elastic 7. Rinse in 3 changes of 70% alcohol bylaminae of arteries, however, stain a brilliant dipping the staining basket 10 times intopurple with this stain. each. S. Rinse in RTW for 5 minutes. 9. Stain in working solution of Weigert's DIsCUssIoN hematoxylin for 10 minutes. Although the individual components of the 10. Rinse in RTW for 10 minutes. technique presented here are well known and 11. Stain in collagen stain A or B for 30 min- utes. tried methods, their successful combination in Ha. If B (Combined collagen stain) is used,the same histological section presents a distinct rinse in 0.25% HC1 by dipping the stainingadvantage. basket into it 5 times. In a study of various collagen staining tech- 12. Dehydrate quickly in 3 changes of absolute ethanol by dipping the basket into eachniques, Constantine and Mowry (5) found that 10 times. none was specific for collagen. They found the 13. Clear in toluene or xylene and mount. picrie acid methods to be the most selective, particularly when acid fuchsin or sirius red RESULTS F3BA were used (7). They noted that while Nuclei are light brown and cytoplasm ligbtsirius red in the picrosirius red stained the yellow. Collagen stains red when stained withfiner collagen fibers more completely, the acid either one of the collagen stains. When picro-fuchsin in the picrofuchsin stained the larger sirius red F3BA is used alone, the large collagencollagen fibers better. Hence, it was reasoned bundles are often incompletely stained as hashere that a combination of the two dyes would been discussed previously (7).Thefine col-give optimal results for staining both large and lagen fibers, particularly those adjacent tofine dermal collagen fibers. This, indeed, is the epithelial structures, stain a brilliant red. Oc-case. The combined picric acid sirius red-acid casionally, keratin of the stratum corneumfuchsin stain is the best collagen stain in the stains red in some areas. On polarization mi-author's experience. The only problem is the croscopy, however, collagen is brilliantly bire-same as is true of acid fuchsin in general. fringent, whereas no other substance stainedNamely, that within a year or so certain with the picrnsirius red becomes birefringent. batches of acid fuehsin have a tendency to fade. When the combined connective tissue stainIn those sections the collagen becomes partly decolorized. This is a distinct disadvantage if 1'Th calculate the actual weight of acid fuchsin, divide 0.03 gin by the per cent of purity indicatedslides are to be kept permanently. However, if on the container. slides are prepared for temporary use and for C0NNCTIVE TISSVE STAIN 355 photography the combined connective tissueing aldehyde fuchsin except that the brown stain is recommended over the picrosirius redelastic fibers do not contrast as well with the F3BA. red collagen. The concentrations of sirius red and acid The acidic carbohydrates are stained with fuchsin may be increased or decreased. SuchMowry's Alcian blue technique (3) with the changes, unless excessive, will affect the in-exception that the staining time has been re- tensity of staining but not the selectivity. Theduced from 2 hours to 30 minutes. Although length of staining may also be varied. Quitethe author agrees with Mowry that 2 hours of good results are obtained by staining for onlystaining is optimal, the difference in intensity 10 minutes, but 30 minutes seems optimal. of staining between specimens stained 30 min- Of the standard techniques for stainingutes and 2 hours is quite small. The 30 minute elastic fibers, Gomori's aldehyde fuchsin is pre-staining time is preferred since for this com- ferred. It shows good contrast with the collagenbined stain the 2 hours staining time is ex- which is stained red. Aldehyde fuchsin does notcessively lengthy. However, for those who pre- stain collagen which has been altered by variousfer it, the 2 hour staining time may be used chemical and physical procedures, whereas theexactly as prescribed by Mowry (3). other elastic tissue stains do (9). The Verhoff Weigert's hematoxylin was found to produce stain for elastic fibers often has a large degree ofgood results with the technique. Harris' hema- non-specific staining of nuclei, stratum corneum,toxylin, which was initially tried, resulted in a and other structures unless the decolorizationreddish color of the nuclei as a result of the process is carried out extensively in which caseacid pH of the collagen-staining mixture. many of the smaller elastic fibers tend to lose The order of the staining procedure is im- their staining. Sweat, et al.(6)have found thatportant. Both Alcian blue and aldehyde resorcin fuchsin is incompatible with sirius redfuchsin are extremely fast stains and either F3BA. The fact that aldehyde fuchsin alsocould be used first. Prior Alcian blue staining, stains some acidic carbohydrates poses nohowever, renders aldehyde fuchsin more highly problem. Previous staining of the sections withselective. The staining of elastic fibers is not Alcian blue blocks almost all of the acidic bind-affected by the Alcian blue presumably because ing sites. For example, the mucin in adenoidelastic tissue staining is on the basis of hy- basal cell carcinomas which has been describeddrogen bonding (12). This is in contrast to to stain with aldehyde fuchsin (10), stainedstaining of acidic carbohydrates, nuclei and with the Alcian blue only. collagen, all of which are primarily on the Acid orcein, initially introduced by Unnabasis of ionic binding. Alcian blue is a basic dye and popularized by Pinkus in his orcein-Giemsawhich combines with the acidic groups of car- stain (11), is also an excellent elastic tissuebohydrates with great tenacity. Whereas many stain. Although it consistently stains nuclearother basic dyes (e.g., hematoxylin, Toluidine, material, the acid orcein stain is otherwiseblue, etc.) can be removed by exposing the highly selective for elastic tissue. Those whosection to a 1% HC1 solution for a few seconds, consider it the best elastic tissue stain mayAlcian blue is not affected. In Mowry's modifi- substitute it (using certified synthetic orcein)cation of the low pH Alcian blue staining for the aldehyde fuchsin stain. Steps 1 throughtechnique for sulfated carbohydrates, the 5 are performed as directed. Then the slidesstained sections are exposed to 0.5 N HCI are stained in 1% orcein and 0.6% HC1 in 70%(about 1.8%) for a total of 9 minutes with no alcohol for 45 minutes (30—60 minutes). A twoappreciable loss of color from the sulfated minute rinse in distilled water, 10 dips in 95%material (13). alcohol, and 5 minutes in absolute alcohol fol- Hematoxylin is a cationic dye which is not low. Then the slides are hydrated and washedvery fast; hence, it is removed by acidic solu- in running tap water for 3 minutes. Followingtions. The collagen staining mixture is acidic; this the connective tissue procedure is resumedtherefore, it removes part of the hematoxylin. starting with step 11. Staining with hematoxylinFor this reason the slides are first overstained is omitted since orcein stains the nuclei. Thewith hematoxylin prior to collagen staining. results are comparable with the procedure us-The picric acid method of collagen staining 356 THEJOURNAL OF INVESTIGATIVE DERMATOLOGY must be the final step prior to dehydration too Division, McGraw-Hill Book so., New York, 1965. since immersion in either water or alcohol for 2. Conn, H. J.: Biologicol Stems. The Williams more than a few seconds will remove the and Wilkins Co., Baltimore, 1961. picric acid and part of the sirius red and acid 3. Mowry, H. W.: The special value of methods that color both acidic and vicinal hydroxyl fuchsin. groups io the histochemical study of mucins. This staining procedure was developed using Ann. N. Y. Aead. Sci., 106: 402, 1963. 4. Comori, C.: Aldehyde-fuchsin: A new stain formalin-fixed human tissues. Animal tissue or for elastic tissue. Amer. J. Clin. Path., 20: human tissues fixed with other fixatives may 665, 1950. not yield similar results and must be evaluated 5. Armed Forces Institute of Pathology: Monuol of Histologic ond Speciol Staining Technws, individually. 2nd ed. The Blakiston Division, McGraw- Hill Book Co., New York, 1960. sUMMARY 6. Sweat, F., Puchtler, H. and Rosenthal, S. I.: Sirius red F3BA as a stain for connective A relatively simple and reproducible histolog- tissue, Arch. Path., 78: 69, 1964. ical procedure is described for the selective 7. Constantine, V. S. and Mowry, H. W.: Se- lective staining of human dermal collagen staining of three main extracellular connective II. The use of picrosirius red F3BA with tissue components in the same histological sec- polarization microscopy. J. Invest. Derm., 50: 414, 1968. tion. Acidic carbohydrates (both connective tis- S. Constantine, V. S. and Mowry, H. W.: Se- sue and epithelial mueins) stain blue with lective staining of human dermal collagen I. Aleian blue, elastic fibers stain purple with al- An analysis of standard methods. J. Invest. Derm., 50: 419, 1968. dehyde fuehsin and collagen stains red with 9. Fullmer, H. M. and Lillie, H. D.: The staining sirius red F3BA with or without the addition of of collagen with elastic tissue stains. J. Histo- acid fuehsin. Mast cells and fungi are promi- chem. Cytochem., 5: 11, 1957. 10. Johnson, W. C. and Helwig, E. B:: Histo- nently stained with Alcian blue with some chemistry of primary and metastatic mucus- additional staining from aldehyde fuchsin. This secreting tumors, Ann. N.Y. Acad. Sci., 106: 794, 1963. procedure is recommended for the histological11. Pinkus, H.: Acid orcein-. Arch. study of connective tissues in formalin-fixed Derm., 49: 355, 1944. human tissues, particularly skin lesions. 12. Goldstein, D. J.: Ionic and non-ionic bonds in staining with special reference to the ac- The author gratefully acknowledges the valu- tion of urea and sodium chloride on the able technical assistance of Mrs. Mary K. Trout. staining of elastic fibers and glycogen. Quart. J. Micr. Sci., 103: 477, 1962. 13. Constantine, V. S. and Mowry, H. W.: Histo- REFERENCES chemical demonstration of sialomucin in 1. Lillie, H. D.: Histopothologic Technic cad human eccrine sweat glands. J. Invest. Derm., Procticol Hiatochemiatry, 3rd ed. The Blakis- 46: 536, 1966.