STUDIES ON NUCLEIC ACID METACHROMASY II. Metachromatic and Orthochromatic Staining by Toluidine Blue of Nucleic Acids in Tissue Sections

Acrolcin-fixed, polyester wax-cmbcddcd tissue sections showed excellent preservation of light microscopic architecture and, when stained with toluidinc blue, intense color contrast between DNA, which stained orthochromatically, and RNA, which stained metachromatically. This method has practical value for differentiating DNA from RNA in the same section. The color c~)ntrast was impaired by substituting formaldehyde for acrolcin or paraffin for polyester wax, and was ncgligible in tissues fixed in formaldehyde or Carnoy's fluid and cmbeddcd in paraffin. Quality of structural preservation paralleled degrcc of color contrast. Mctachromatic staining can be analysed, by the quantitative parameters of Bradley and colleagues, to provide inferences regarding the conformation of biopolymers in tissue sections. Comparison of the nucleic acid color contrasts in toluidine bluc-stained sections with titrations of fixative-treated nuclcic acids against toluidine blue in solution indicated a grcater difference in conformation betwecn DNAand RNA-protein in acroleinpolyester sections than between acrolein-trcated frce DNA and RNA in solution. This is supported by recent cvidencc that the conformation of ribosomal RNA is quite different in whole ribosomes from that assumed by the same RNA free in solution. The acrolcinpolyester method may enhance color contrast by providing superior preservation of ordered nucleoprotein conformations. I N T R O D U C T I O N While studying the properties of a new fixative, acrolein, we noticed that tissue specimens fixed in this agent, embedded in polyester wax (20, 21), and stained with the metachromatic dye, toluidine blue, showed a striking color contrast between structures containing DNA, a which were stained orthochromatically (blue-green), and i Abbreviations employed : DNA, deoxyribonucleic acid; RNA, ribonucleic acid; P/D, the ratio of the number of polymer dye-blnding sites to the number of dye molecules in any given polymer-dye system; K, stacking coefficient. structures containing RNA, which were stained metachromatically (purple). Although similar color contrasts have been described elsewhere (8, 18), the consistency and magnitude of the color contrast in acrolein-polyester wax sections surpassed anything obtained with our toluidine blue staining procedure after other preparative methods. This method appeared to have considerable practical value for distinguishing DNA and RNA in tissue sections. Furthermore, the metachromasy of polyelectrolytes is known to reflect certain features of their molecular con327 on A uust 4, 2017 jcb.rress.org D ow nladed fom f o r m a t i o n in a q u a n t i t a t i v e fash ion (3-5) . A m e t a c h r o m a t i c color c o n t r a s t b e t w e e n D N A a n d R N A m i g h t offer c lues to the i r c o n f o r m a t i o n in t issue sections. W e h a v e therefore inves t iga t ed t h e re la t ive c o n t r i b u t i o n s of t h e f ixat ive a n d the e m b e d d i n g m e t h o d to the color c o n t r a s t in ac rol e in -po lyes te r sect ions. T h e resul ts a re d iscussed in t e r m s of t h e s p e c t r o p h o t o m e t r i c t i t ra t ions desc r ibed in the p rev ious p a p e r (14). M A T E R I A L S A N D M E T H O D S Smal l pieces ( m a x i m u m size 5 m m thick) of brain, tongue, d u o d e n u m , liver, pancreas , spleen, testis, a n d epididymis of adul t ra t and ribs of newborn ra t were fixed and embedded . Some specimens were fixed in acrolein; some in formaldehyde, the s t andard a ldehyde fixative; and some in Carnoy ' s fluid, a simple d e n a t u r a n t fixative c o m m o n l y employed in nucleic acid studies. Acrolein was used as a 10 per cent solution in 0.5 per cent aqueous ca lc ium acetate (15). Similar results have been obta ined with 10 per cent acrolein in i / 1 0 phospha te buffer at p H 7.2, in Tyrode ' s ba lanced salt solution, in p la in water, or in xylene. Fo rma ldehyde was used as 10 per cent formalin (i.e., final concent ra t ion 3.8 per cent fornlaldehyde) in 0.5 per cent aqueous ca lc ium acetate. Carnoy ' s fluid is 10 per cent (v/v) glacial acetic acid, 60 per cent ethanol , and 30 per cent, chloroform. Th i s fo rmula t ion was chosen because of its previous use by Schf immelfeder et al. (19) in studies of acridine orange metachromasy . W i t h acrolein, o p t i m u m fixation and color contrast were achieved after fixat ion for 1 h o u r at r o o m tempera tu re or overnight at 0°C. Wi th formaldehyde, the o p t i m u m t ime was overnight at r o o m tempera tu re or several days at 0°C. Fixat ion in Ca rnoy ' s fluid was carr ied out at r o o m tempera tu re for 4 hours. A few specimens were first fixed in one and then postfixed in ano ther of the solutions. Paraffin wax was c o m p a r e d with polyester wax. Some specimens fixed in each way were dehydra ted in g raded ethanols, t ransferred to xylene, and embedded in paraffin wax. O the r specimens were dehydra ted in a 1:1 mixture of m e t h a n o l and methoxye thanol (7), t ransferred to e thanol and then to n-propanol , and e m b e d d e d in polyester wax (20, 21). O the r me thods of dehydrat ion, preceding e m b e d d i n g in polyester wax, gave similar results. Sections were cut at 5 /z, m o u n t e d on glass, dewaxed, hydra ted , s tained for 8 minu tes in aqueous toluidine blue in 0.02 M benzoate buffer, p H 4.2 (21), dehydra t ed in tert iary butyl alcohol for 5 minutes , as r e c o m m e n d e d by Michaelis (17 a) and Flax and Himes (8), t ransferred to xylene, and m o u n t e d in Pcrmount . A toluidine blue s ta ining solution buffered with phospha te gave similar results. Toluid ine blue 0, Color Index 52040, was used as purchased. In addition, a few slides were s ta ined with a sample of to lu id ine blue purified and supplied by L a m m , Childers, and Wolf (14). The same results were ob ta ined with bo th samples of dye. F lax and Himes (8) r e c o m m e n d azure B over o ther me tach roma t i c dyes for differential s ta in ing of nu cleic acids. In our hands , toluidine blue 0 proved equal or slightly superior to azure B for this purpose, whe the r s ta ining was per formed at r oom t empera ture (22°C) or at 40°C as done by Flax and H imes (8) and Pelling (18). Sta ining at the h igher t empera ture gave a greater color contrast , par t icular ly with Carnoy 's-f ixed mater ia l ; bu t the results of s ta ining sections at r oom tempera tu re can be directly correlated with the spect rophotometr ic t i t rat ions described in the c o m p a n i o n paper , since these were carr ied out at r oom tempera tu re of 22°C. The descr ipt ion and discussion tha t follow are therefore based exclusively on s ta ining at r oom tempera ture . The color of D N A was s tudied in vegetat ive nuclei, in sperm heads, and in the ch romosomes of dividing cells. T h e color of R N A was s tudied in nucleoli, in the zone between separa t ing te lophase chromosomes of d ividing cells (12), and in the ergastoplaslTl of cells such as neurons, liver pa r enchyma l cells, and pancreatic ac inar cells. T h e color contrast be tween D N A and RNA-r i ch s t ructures was visually es t imated as 0 to -[+ -]+ in te rms of a rough scale of increasingly me tach roma t i c color: b lue-green th rough blue to purple. Thus , 0 indicates identical colors; +,+ the smallest contrast tha t could be positively identified by both authors ; -{--}-, a larger contras t such as between blue and purpl i sh blue or be tween blue-green and blue; and + -}+ + , the largest contrast observed, tha t be tween blue-green and purple. T h e acrolein-polyester method , in addi t ion to its effect on nucleic acid hues, strikingly enhanced the me tach romasy of s t ructures conta in ing acid mueopolysaccharides, such as cart i lage matr ix , the granules of blood and tissue basophils, and mucous droplets in goblet cells. This effect will be the subject of a separate report. T h e color contrasts p roved difficult to record in color photographs . Satisfactory results were finally obta ined with Ek tach rome Type B 4 X 5-inch sheet fi lm th rough the use of exposure t imes and color compensa t ing filters individual ly selected by trial and error for each new ba tch of fihn. Evaluat ions of s t ructura l preservat ion were based on lifelike cont igui ty of cells and extracel lular components, wi thout artificial shr inkage spaces; on predominan t ly smooth and cont inuous outlines of cells and nuclei; and on delicacy of detail in nuc leoplasm and cytoplasm. Parallel sections were s tained with acid fuchsin to assess the s t ructura l integri ty and stainabil i ty of mitochondria . 328 THE JOURNAL OF CELL BIOLOGY • VOLUME ~7, 1965 on A uust 4, 2017 jcb.rress.org D ow nladed fom