SEPARATION O F THE ACIDOPHILIC ELEMENTS OE' THE TISSUES INTO TWO GXOL'PS The prol'ouncliy differential staining reactions obtained by the use of phosphotu~~gsticand phorphomolyhdic acid i n corinectioii with certain acid dyes have long been knoma. AS applied for example i n tlie well-known Xallory 's connective tissue stain, the method sen'es to divide the cytoplasmic elements of mammalian tissue into t w o major groups according t o their staining reactions. It consists in staining Z e n k e r - h c d material in acid fuclisin, which confers a more o r less uniform color upon the tissue. Sections a r e transferred t o pliosphomolylsclic or phosphotungstic acid f o r selective differentiation. (lertain tissue elements, relatively acidophilic, including general cytoplasm, muscle, erythrocytes, acidophil cells of the pituitary, epmogen (~7hichwe may call claw 1) retain tlie acid fuchsin ; others, iiicludiiig collagen, reticulum, cartilage, niucin, basophil cells of tlie pituitary (class a), a r e completely decolorized. Since it is desirahle that these elements 1iax.e color, a secondary dye, anilin blue, is used f o r iliat purpose. Various modifications of Mallory's original stain employing other fixatives and dyes, when intelligeiitly performed and microscopically controlled, >-ield similar results with respect t o the staining reactions briefly described above, Orange G may be incorporated with the secondary dye, milin blue (Mallorp, '00) or with the primary dye, acid fuchsin (Crossmon, '37). If material is fixed in Zenker's or a bichromute fixative, erythrocytes possess a greater affinity for orange G than acid fuchsiii and consequently a r c I(i3 164 GERJZIIIN c. C11OSS3IOF stained orange. The application of orange G has h o n w e r no bearing 011 the following discussion, and no further 1-cfereiice need be niade to this component of RLallory's stain. The specific action of pliospliotungstic acid npoii certain elements of the tissues is well illustratecl by the use of Congo red. The dye acid is blue, but its sodium salt is red. The red color of the salt is readily changed by weak acids into blue. If sections a r e stained with a saturated solution of this dye containing R small amount of sodium chloride, the tissue is quite homogeneously stained red. Sections a r e transferred to 2% phosphotungstic acid f o r a few seconds, qriicldy rinsed in distilled water, and examined under the microscope. Tissue elements of class 1 a r e red, those of class 2 a r e blue. Phosphotungstic acid in aqueous solutioii is unable to remove this dye selectirely from the secondary elements pet expresses its specificity by changing their color to blue. The result is a Mallory stain with the employmelit of one dye. Permaiient preparations i n xylol balsam a r e however not possible brcause the blue coloration of the secondary clemcnts is gradually discharged in ethyl alcohol. Examination of sections stained 1)p Shllory 's original method sliows that certaiii components forming class 1 are more intensely stained than others, because these more acidophil elements have a greater affinit~7for acid fuelisin. This is also true when certain other dyes a r e iisecl as the primary stain, e.g., poncenu tlc xylicliiie (Masson, '29). Preparations stained with hematoxyliii and eosiii show this same tinetorial differentiation. A few of these elements long since observed to have greater affiriitv f o r eosin a n d consequently t o be more intensely stained by it have been termed eosinophilic, o r more generallj-, acidophilic or osyphilic. It occurred to the Tvriter that with appropriate technique and choice of dyes these moi*e intensely staining acidophil elements of class 1 might be niorc sharply iliffereiitiated. Experiment with rarious acid clyeq showed that they may be divided illto three groups accordiiiq t o their reactions wit11 phosphotungstic acid. 165 SEPARATION O F A C I D O P H I T J C ELENEXTS 1. Dyes that show no reaction to phosphotungstic acid. Mercurochrome is the oiilj7 example arailable at this time. 2. Dyes such as acid fuchsin or ponceau de xylidine that may be used for. the division of liistological elenleiits into two major classes. 3. Dyes that hare affinity f o r only the strongly acidophil elements of protoplasm. The fluorescein dyes eosin Y, erythrosin, phloxiiie and rose beiigal are i n this category. Chromotrope 2R, an azo dye closely relatcd to orange G, is also in this group, hut its usc has not yet been fully investigated. With this information, ihe following modification of Xallory’s stain, cmploying the iluoresceiii dyes in idace of acitl fuchsin, has been devised. 1. Fixation. The usual methods of fixation may he einployed. Bichromate-f orrnaliii (or Zenkcr-formaliii) is essential f o r the preservation of zj-mogen granules or other histological elemeiits soluble in acid fixativcs. Bouin’s fluid is useful for the demonstration of erythrocytes and othw aciclopliilic clcrnents riot sohnbl e in acid fixatives, a i d when used particularly f o r erythrocytes has the adrantage that maiiy other strongly stainable siriall elements (e.g., zymogen granules) are not pi~escrvccl. Neutral 10% formalin containing 0.85 gni. of sodium cliloride f o r each 100 cc. is also a suitable general fixative. The fixing finids should be prepared and used exactly a s follows. Rnclwoniate formolui (proportions as used by Masson, ’29) 3 % potassium bichromate in distilled water Comincrcial f orinalin (neutralized) 90 cc. 1 0 cc. Commercial formalin map be neutralized by the addition of niarble chips a fern days prior to fixation. The mixture is relatively unstable and should be p~epareclimmediately before use, The duration of fixation is 18 to 24 houi*s,according to the size of material. Pieces should not h s greater than 8 mm. in 166 GEBMAIN C. CROSSMON thickness j their other dimensions are of no importance. The quantity of fixing fluid should be a t least fifty times the volume of the tissue. Precipitation gradually occiirs in the bichromate formalin mixture after a few hours. Pieces should be transferred to freshly prepared fixative. One or two changes are usually sufficient. Following fixation, material is washed in running tap water for 18 t o 24 hours and transferred to 70% ethyl alcohol, preliminary to further dehydration. Houin’s fluid Picric acid, saturated aqueous solution Commercial formalin Glacial acetic acid 7 I ,> c r . 20 cc. 5 cc. This mixture is stable and may be prepared in large quantities. The size of pieces and time of fixation are as f o r bichromate formalin. Following fixation, material is washed in 70% ethyl alcohol. Several changes should be used to remove excess picric acid. 2. Dehydrate completely, embed in paraffin, cut and mount as usual. 3. Stain in Mayer’s acid liemalniil (standard formula as given in Lee’s Vade-Xecum). The nuclei should be slightly overs t ained. 4. Wash sections in running tap water €or at least 15 minutes followed by a change to distilled water. 5. Stain in thymol erythrosin. Erythrosin, bluish (National A oiline Co.) Thymol ergatal Distilled water 0.5 gm. 0.1 gm. 500 re. Heat gently until thyniol and erythrosin are dissolved. Tlic solution should be filtered, as a slight precipitate usually results. The addition of thynzol is not necessary but of value because it increases the intensity of staining. In this laboratory it is always used with eosin P wlienever that dye is used as an acid counterstain, f o r example with hematoxylin. SEPARATTOK O F AClDOPEIlLlC ELERTEKTS 167 Other fluorescein dyes iiiclnding eosiii 37, phlosine a n d rose bengal may be substituted for erythrosin in the formula above. As stated above, niercurochromc is an exception j phosphotuiigstic acid shows no selective action with reference t o this fluorcsceiii dye. A suggested staining time is 20 minutes. Somewhat morc‘ intense results may be obtained by staining for a longer interval. 6. Rinse i n distilled watcr. This sicp is sometimes omitted. It is nsually necessary if tlie scctioiis contain striated muscle. 7. Transfer to 2% phosphotungstic acid i n distilled water. Sections a r e left in this iaeagent 15 to 20 minutes or until the elements of class 2 a r e microscopically orange, components in class 1remaining red. Like Congo red, erytlirosin is an indicator. Phosphotungstic acid in aqueous solution is unable to remove the dye from cornpoiients of class 2 yet expresses its specificity hy changing their color. F o r best results fresh phosphotungstic acid should he used f o r every second rack of slides. 8. Rinse in distilled water. This step is sometimes omitted. It is usually necessary if sections contain striated muscle. 9. Transfer t o absolute ethyl alcohol. Seetions shoulcl be gently agitated to allow for imiform and rapid decolorization. Elements of class 2, colored orange in step 7, a r e immediately decolorized. Smooth rnuscle, skeletal muscle and general cytoplasm, colored red in step 7, require a longer time. As a final result, erythrosin is removed from all tissue elements with the exception of thosc histological structurcs that arc strongly acidophil. Sometimes a little difficulty niay be experienced i n removing all dye from muscle substance, especially with thick o r torn scctions. If this occurs, slides a r e transferred from the absolute alcohol back to phosphotnngstic acid for a n additional time interval. A t this stage the strongly acidopliilic elements are red, tlic less strongly aeidophilic and the basophilic elements are colorless, and the nuclei have been stained by the hematoxylin. 168 G EI t M A I N C . CROSSINOF The procedure limy be terminated by coinpleting dehydration in one or more clianges of absolute alcohol, follomd by clearing in xylol and mounting in xylol balsam. It will however usually be desirable t o introduce color into the elements left colorless by the preceding steps. Proceed as follows. 10. Reniordaiit in 2% phospliotungstic acid (repeating step 7) f o r 2 to 3 minutes. 11. Transfer to light green (or aiiilin blue). liiglrt green (Iira11 Microcolor ; Eiiiicr :tncl L l ~ l ~ ( ~ n d ) Distillcd water Glacial acetic acid 1 gm. 100 w. 1 cc. Since it is dificnlt t o examine the preparation in this stain, the snggestccl time is 2 to 4 minntm. Light green and aiiiliii blue are mordanteil to the elements of class 2. With additional staining time the decolorized elements of class 1 map be lightly stained. The w e of' light green is suggcstecl for most preparations. F o r the demonstration of the basophil cells of the pituitaq-, iiiucin and certain other basophil structures, anilin blue is superior. The formula is : Aiiilin blue (Xational -\iuline C'o.) I)istilld water Glacial avrtic acid 2 gm. 100 ec. 2 cc. The procedure is practically the same as with light green, except that sections are left comparatively longer in the glacial acetic acid (step 13) t o obtain optimum dilferentiation. 12. Rinse in clistilled water, renioring the sectioris inimecliat ely. 13. Traiisl'cr to 1% glacial acetic acid to remove the light green or aiiiliii blue looscly bouiid to other than elements of class 2. Sections shoiild be left in the acid until that result is attained. Iiispection of a trial slide with the microscope will determine optimum dillerentiation. Thirty to 60 seconds is usually sufficient for light green. SEPARATION 04’ AClDOI’HlLiC 169 ELE3IES TS The final result should he as indicated in the table at the end of this paper. Those elements listed in class 1A still retain the erythrosiri or other substituted fluorescein dye. Those listed as class IB, having little aEnity for either primary or secondary dye are colorless or very lightly stained by the light green or aiiilin blue. 14. Rinse in distilled water removing the sections immediately. 15. Pass througli two or three changes of absolute ethyl alcohol followed by three changes of sylol. Mount in balsam. UISCOS8ION Results obtained, at this time of writing, with respect to color are listed in the following table. Class 9 Glusa 1 9 R Red Colorless o r vei11 rightly ntauied , q > c e n or blii? ( i r e m or blur General cytoplasm Smooth muscle Collagenous fibers Reticulum Erythrocytes Eosinophil granules of leucocytes Certain zyniogen granules Russell bodies of carci noma, sarcoma Acidophil cells of the pituitary Xeratinized epithelium Skrletal musclr Cartilage EWll2 Dentine Basophil cells of pituitary The staining reactions of the components of class 2 mill be well known to the reader through the use of the original Xallory’s stain. The separation into two classes, of the elements staining with the acid fuchsin in the original Jlallory’s stain, which is achieved by the new technique herewith described, will require further study. Alan)- questions suggest themselves as to chemical reasons f o r the high affinity for acid dyes shown by the elenieiits of class l A , and also as l o possible relationship (or lack of relation) kd,weeri these elements, seemingly so diverse. 170 GERMAIN C. CROSSMON Specific applications are also a question for additional consideration. Other workers may firid that the Substitution of other fluorescein dyes o r other methods of fixation serve better a particiilav purpose. The time of staining or ileeolorization should be considered as open to modification to suit specific cases. LITEBATURE crrm G. c. 1937 A modification of iVallorp’s connective tissue stain with a discussion of the principles involved. Anat. Ree., vol. 69, pp. 33-38. MALLORY,F. B. 1900 A contribution t o staining methods. J. Esp. hled., rol. 5, pp. 15-20. MASSON, P. 1939 Some histological methods. Triclirome stainings and their CXOSSMON, preliminary technique. . I . Tech. Methods, >ol. 12, pp. 75-90.