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Bismuth Hematoxyh Stain for Arginine Residues
John B. Roach, Jr. and Allen A. Smith School of Podiatric Medicine, Barry University, Miami Shores, Florida 33 7 6 7
ABSTRACT. Bismuth ions complex with hema- toxylin oxidized by sodium iodate to form a dark blue dye that stains structures with high arginine content. In citrate buffer at pH 5.2, staining is confined to cell nuclei and myelin sheaths. Ex- traction of nucleic acids has little effect on the stain. Blockade of the guanidino groups of argi- nine completely abolishes staining.
Key words: arginine, bismuth, hematoxylin, his- tones, myelin
ematoxylin was introduced into histology H in the 17th century (Sheehan and Hrap- chak 1980) and remains a popular histological stain because of the intensity and permanence of the stain. Its usefulness is enhanced by its versatility. Hematoxylin is bound to the tissue by a mordant to display the tissue components to which the mordant binds. The nature of the mordant determines which tissue components are displayed by hematoxylin staining (Smith 1995a).
The specific staining of basic proteins by base-differentiated vanadate hematoxylin (Smith 1995a) led us to search for a mordant that would bind hematoxylin to certain basic proteins allowing us to develop a specific stain for histones. Hayat (1981, 1993) suggested that Albersheim and Killias’ ( 1963) citrate-buffered bismuth electron stain for chromatin actually binds to the arginine residues of histones. This led us to develop a combination of bismuth and hematoxylin that would stain arginine residues with sufficient intensity for light microscopy.
Most of the present study was performed us- ing the citrate buffer of Albersheim and Killias
Address correspondence to: Dr. Allen A. Smith, School of Podiatric Medicine, Wiegand Hall, Barry University, 1 1 300 NE 2nd Ave., Miami Shores, FL 33 161
1052-0295/97/49-54/$3.00 BlOTECHNlC & HISTOCHEMISTRY Copyright 0 1997 by Williams & Wilkins
Volume 72 Number I
(1963) because their stain was more selective for chromatin than the tartrate-triethanola- mine buffered stain of Locke and Huie (1977).
MATERIALS AND METHODS A white mouse was killed by cervical disloca-
tion. Pieces of tongue, larynx, pancreas and cer- ebellum were fixed for 6 days in 10% formalin or 3 days in Bouin’s fluid (Carolina Biological Supply Co., Burlington, N.C.), dehydrated in al- cohols, cleared in xylene and embedded in “Par- aplast” (Shenvood Medical, St. Louis, MO). Sec- tions were cut a t 8 pm, mounted on glass slides and warmed a t 55 C for 48 hr.
Hematoxylin (EM Science, Gibbstown, N J ) was dissolved in 5 ml of ethanol and diluted to 50 ml with buffer and glycerol. Bismuth nitrate pentahydrate ( Bi(N03),.5H20) and sodium io- date (NaIO,) or sodium bismuthate (NaBiO,) were added and the mixture stirred for 20 min. The pH was measured with a “Checker 1” pH tester (Hanna Instruments. Woonsocket, R.I.].
We tested many variations of these staining solutions. The best histone staining was ob- tained by dissolving 50 mg bismuth nitrate pen- tahydrate in 85 mlO.1 M citric acid and adding 10 ml glycerol and 19 ml 1 M sodium hydroxide. After adjusting the pH to 5.2, 50 mg hematoxy- lin in 5 ml ethanol was added followed by 10 mg sodium iodate in 1 ml water. This solution should be stirred for 20 min before adding a n additional 10 ml glycerol. The staining solution has a shelf life of 3 hr. Sections were stained for 3-30 min, but the optimal staining time was 10 min.
DNA and RNA were extracted from some sec- tions by immersing them in 4% trichloroacetic acid for 15 min at 90 C or in 5% perchloric acid for 30 min at 60 C (Kiernan 1990).
The amino groups of basic amino acid resi- dues were blocked in some sections by immers- ing them in a solution of 10 ml acetic anhydride
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50 Biotechnic & H istochemistry
and 0.1 ml sulfuric acid in 30 ml glacial acetic acid (Kiernan 1990).
Lysine residues were deaminated in some sections by immersing them in a solution of 3.5 g sodium nitrite (NaN02) and 3 ml glacial acetic acid in 50 ml water for 24 hr at room tempera- ture (Kiernan 1990).
The guanidino groups of arginine residues were blocked in some sections by imidazoliza- tion using a solution of 1.6 g benzil, and 0.8 g sodium hydroxide in 80% ethanol for 1 hr at room temperature (Kiernan 1990).
RESULTS At pH 5.2, bismuth hematoxylin stains his-
tones strongly and myelin basic protein weakly in tissues fixed in Bouin's fluid. It does not stain collagen (Fig. 11, erythrocytes (Fig. 2), muscle proteins (Fig. 3) or cartilage (Fig. 4). The rough endoplasmic reticulum of pancreatic acinar cells (Fig. 2) and the Nissl substance of neurons
Fig. 2. Mouse pancreas fixed in Bouin's fluid and stained with bismuth hematoxylin at pH 5.2. Nuclei of acinar cells stain, but rough endoplasmic reticulum (arrows) does not. Erythrocytes in the venule at upper right do not stain. The secretion product in the duct below the venule does not stain. X 500.
Fig. 1. Cortex of mouse lymph node fixed in Bouin's fluid and stained with bismuth hematoxylin at pH 5.2. Capsular collagen (arrow) does not stain. The valve of a lymphatic vessel is at right center and part of an artery is at lower left. X 125.
[Fig. 5) did not stain at pH'5.2. I t stains kerato- hyalin strongly and keratin weakly (Fig. 3). The nuclei of chondrocytes are stained weakly (Fig. 4) while all other nuclei are stained strongly (Figs. 1-31.
Staining for 5 min produces a pale stain while 10 min gives a dark nuclear stain. Longer stain- ing does not improve the stain.
Tissues fixed in l , O % formalin stain poorly with bismuth hematoxylin.
The bismuth hematoxylin solution described here does not stain if sodium iodate is omitted, even if hematein is substituted for hematoxylin. Substitution of sodium bismuthate for the bis- muth nitrate and sodium iodate gave equally good staining, but the shelf life of the solution is shorter.
Using more than 50 mg of bismuth nitrate pentahydrate in 120 ml of staining solution causes some staining of rough endoplasmic re- ticulum; using less than 50 mg weakens nu- clear staining. Less than 50 mg of hematoxylin
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Bismuth Hematoxyl in 51
, .1 . ’
Fig. 3. Mouse tongue fixed in Bouin’s fluid and stained with bismuth hematoxylin at pH 5.2. In addition to cell nuclei, keratohyalin (arrows) i s stained. x 125.
in 120 ml of staining solution weakens the stain while using more than 50 mg results in a slight loss of specificity.
Omitting or decreasing the quantity of glyc- erol shortens the life of the staining solution, but increasing the quantity of glycerol above 20 ml does not lengthen it. Increasing the quantity of sodium iodate markedly shortens the useful life of the staining solution.
Bismuth hematoxylin in citrate buffer stains both the Nissl substance and rough endoplas- mic reticulum in weakly to moderately alkaline solutions (pH 8-1 1). It fails to stain in strongly alkaline (> pH 12) or strongly acidic (c pH 3) solutions.
The life of the stain is shortened to a few min- utes when the staining solution is made with water instead of buffer, and this has led to in- correct comparisons of experimental and con- trol tissues (Smith 1995b).
Substituting tartrate buffer (0.04 M. pH 5.0) for the citrate buffer shortens the life of the stain
Fig. 4. Mouse laryngeal cartilage fixed in Bouin’s fluid and stained with bismuth hematoxylin at pH 5.2. Chondrocyte nuclei (arrows) stain weakly. Surrounding cartilage matrix and collagen (open arrowheads) do not stain. x 125.
to 30 min and causes staining of the Nissl sub- stance and rough endoplasmic reticulum. Al- though bismuth hematoxylin stains both nuclei and rough endoplasmic reticulum in 0 .1 M HEPES (4-(2-hydroxyethyl)-piperazine- 1 -eth- anesulfonic acid) buffer at alkaline pH. it does not stain tissue sections in HEPES at pH 5.0.
Extracting nucleic acids with trichloroacetic acid slightly increased the intensity of nuclear staining with bismuth hematoxylin (Fig. 6). Al- though extracting nucleic acids with perchloric acid had little or no effect on nuclear staining, it did produce Nissl substance and rough endo- plasmic reticulum staining.
Blocking the basic amino acid residues with acetic anhydride and sulfuric acid abolished bismuth hematoxylin staining. Deamination of the free amino groups by diazotization markedly decreased staining. Diazotization had a greater effect on bismuth hematoxylin staining at pH 10.7 than a t pH 5.2. Blockingguanidino groups by imidazolization with benzil completely abol- ished staining (Fig. 7).
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52 Biotechnic & Histochemistry
Fig. 5. Mouse cerebellum fixed in Bouin’s fluid and stained with bismuth hernatoxylin at ptl 5 .2 . Nuclei of Purkinje cells and granule cells (curved arrows) stain while the Nissl substance of Purkinje cells (open arrowheads) does not. X 800.
DISCUSSION The staining of nuclei after extracting nucleic
acids shows that bismuth hematoxylin does not stain nucleic acids. Imidazolylation of the gua- nidino groups of arginine by alkaline benzil completely abolished bismuth hematoxylin staining demonstrating that bismuth hematox- ylin is a specific stain for arginine residues at pH 5.2.
The partial prevention of staining by deami- nation suggests that deamination does affect both guanidino groups of arginine and the amino groups of lysine (Lillie et al. 1971 I. Deam- ination has a greater effect on staining at pH 10.7 indicating that lysine residues participate in the binding of bismuth hematoxylin at alka- line pH.
The complete blockade of bismuth hematoxy- lin staining by acylation of the amino and gua- nidino groups of basic amino acid residues also suggests that one of these groups is responsibIe for bismuth hematoxylin staining. There is some dispute whether the acylation is an ace-
tylation (Lillie 1964, Pearse 1968) or a sulfation (Kiernan 19901, bu t the effect would be the same in either case.
Myelin basic protein is 15% arginine by weight (Lees and Brostoff 1984) while the his- tone proteins of cell nuclei are 12-14% arginine by weight (West et al. 1966, Orten and Neuhaus 19.75). Few other proteins have more than 8% arginine. Bismuth hematoxylin staining was limited to histones and rnyelin basic protein suggesting that the minimum arginine content for staining is approximately 12%. I t is probable that much of the arginine is complexed with other molecules so that proteins with less than 12% arginine content have too few guanidino groups to bind a visible quantity of the stain. Perchloric acid may render the Nissl substance and rough endoplasmic reticulum stainable by exposing guanidino groups on the ribosomal proteins as it removes the RNA. Ribosomal pro- teins are 8% arginine by weight (West et al. 19661.
The staining of keratohyalin and keratin (3% arginine) may be due to the same van der Waals
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Bismuth Hematoxylin 5 3
Fig. 6. Mouse cerebellum fixed in Boulin’s fluid. Nucleic acids extracted with 4% trichloroacetic acid for 20 min at 90 C. Bismuth hematoxylin at pH 5.2. x 800.
effect that causes them to stain with iron hema- toxylin and alum hematoxylin (Lillie et al. 1976, Horobin 19881. Aside from this aberration, bis- muth hematoxylin appears to be specific for ar- ginine residues.
Bouin’s fluid may facilitate bismuth hematox- ylin staining by breaking down the DNA-histone complex. The poor staining of chondrocyte nu- clei may be due to poor penetration of the fixative.
The superiority of citrate to other buffer salts is probably due to its ability to chelate bismuth ions (Considine and Considine 1984).
The inability of hematein to stain nuclei in the presence of bismuth nitrate alone suggests that the mordant is not Bi3+. The ability of hema- toxylin to stain nuclei in the presence of sodium bismuthate alone suggests that the mordant could be Bi5+ or Bi03-. Since hematoxylin stains nuclei in the presence of bismuth nitrate and sodium iodate, it seems IikeIy that iodate is ca- pable of converting bismuth (111) to bismuth (V) in spite of the considerable disparity in redox potentials.
The bismuth (V) in bismuth hematoxylin is likely analogous to phosphorus (V) in a phos- phate ion. In weakly basic solutions, the nega-
Fig. 7. Mouse cerebellum fixed in Bouin’s fluid. Cuanidino group of arginine blocked by 1 hr treatment with alkaline benzil. Purkinje cell nuclei (curved arrows) are unstained. Bismuth hematoxylin at pH 5.2. X 800.
tively charged bismuth hematoxylin can bind to positively charged amino groups through an ionic interaction. In strongly basic solutions (pH > 121, the amino groups have lost their posi- tive charges. In acidic solutions, the bismuth hematoxylin loses its negative charge and can- not bind ionically to amino groups. We suggest that bismuth hematoxylin in acidic solutions imitates phosphate and binds to the guanidino group of arginine as shown below.
OH
/ C=C / - \
n \ q-CH2- cII1--CHz--Nil
/ LH
Bismuth hematoxylin is a reasonably selec- tive stain for histones. Unlike the Sakaguchi reaction (Pearse 19681, it is permanent. Unlike the Biebrich scarlet and fast green methods (Kiernan 19901, it does not require prior re-
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54 Biotechnic & Histochemistry
moval of DNA. Because it does not expose the tissue to strong alkali or acids, bismuth hema- toxylin preserves cellular detail better than al- ternative methods.
Ingested bismuth salts are very poorly ab- sorbed and are not toxic to adults. The nitrate in ingested bismuth nitrate is probably more toxic to infants than the b i s m u t h (Gosselin et al. 1976). Since the environmental toxicity of bismuth is generally believed to be low (Brister 1993, Sautter 19951, disposal of the staining solution is not a serious problem.
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Brister, B. 1993. The three B’s: big-bores, back- bores, and bismuth. Field and Stream 97(3):
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