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Home , Methyl violet, Potassium permanganate

Okajimas Folia Anat. Jpn., 57(6) : 369-386, March 1981

Oxidation-Induced Basophilia of Various Tissues -A Histochemical Comparison of Pre- Oxidative Treatments-

By

MASUMI AKITA

First Department of Anatomy, Saitama Medical School, Saitama 350-04, Japan (Director : Prof. Dr. Katsuji Kaneko)

-Received for Publication, August 7, 1980-

Key words: Basophilia, , Basic

Summary : The significance of increased basophilia in various tissues after treatment with oxidizing agents which are widely used in histochemical studies, was investigated. treatment resulted in a marked increase in basophilia in gastric chief cells, gastric mucin, intestinal goblet cell mucin, thyroidal colloid, elastic fibers, pancreatic acinar cells and B cells, Purkinje cells, hypophyseal neurosecretory substances, and pilary cortex. Performic and peracetic treatment exerted a similar effect on neurosecretory substances, pancreatic B cells and pilary cortex, while periodic acid treatment did so only on pilary cortex. Potassium permanganate treatment resulted in increased basophilia on synthetic polypeptides of , tyrosine and tryptophan, while treatment with performic or peracetic acid exerted a similar effect on synthetic polypeptides of cysteine and tryptophan. The present model tissue experiments and the reaction of chemical end groups of amino following oxidation suggest that the permanganate-induced basophilia of tissue proteins may be attributable to cysteine, cystine, tyrosine, or tryptophan; the per- formic and peracetic acid-induced basophilia to cysteine, cystine or tryptophan ; and the periodic acid-induced basophilia to cysteine or cystine.

Introduction the affinity for basic (toluidine blue and ) in the keratinized Treatment of sections with a strongly portions of epidermis and hair, purified oxidizing agent prior to staining, induces fibrin, cytoplasm of smooth and striated strong basophilia on many cell constitu- muscle fibers, endothelium, and the cells ents and tissue components. Bignardi of pancreatic islets and acini. According (1939, 1940a, 1940b) noted that mucoid to Lillie et al. (1954), keratinous struc- secretion, glycogen and starch exhibit tures (hair shafts, nails, and stratum increased basophilia after treatment with corneum of the epidermis) were stained chromic acid, persulfate or periodic acid. by azure A and thionine after oxidation Dempsey et al. (1950) reported that oxi- with peracetic acid, in carbon dation with periodic acid greatly enhances tetrachloride, or neutral permanganate.

369 370 M. Akita

In the small intestine, oxidation with fixed in 10% formol, Zenker-formol or potassium permanganate or chromic acid, Bouin's fluid, embedded in paraffin and resulted in a small increase in -fast sectioned at a thickness of 5 ,am. metachromasia ; however, the distribution Model tissues were prepared according of the metachromatic areas was patchy to the specifications of Cooper (1971). and the results were too variable to be Synthetic polypeptides (30 mg) were indi- of practical value (Kramer and Windrum, vidually dissolved in 1 ml of 0.1 N aqueous 1954). Glycogen, Brunner's gland mucin and the resultant solu- and certain other substances exhibited tion was added to 1 ml of liquefied 40% increased basophilia with periodic-chromic aqueous gelatin. After cooling (at room acid oxidation (Mowry, 1956). Further- temperature) to a gel, the preparations more, Lison noted that elastic fibers and were briefly hardened in 4% formol and eosinophilic leucocytes were rendered processed for paraffin sectioning. All by potassium permanganate synthetic polypeptides were obtained from oxidation (Gabe, 1976), and renal juxta- Sigma. The synthetic polypeptides used glomerular cell granules were stained by in this study were as follows : ethyl violet (Smith, 1966) methyl violet 1) Poly-L-arginine 2B and (Harada, 1970). (approx. mol. wt. 60,000) These oxidation-induced basophilic tis- 2) Poly-S-benzyl-L-cysteine sue components have been attributed to (approx.mol. wt. 7,700) (Bignardi, 1939, 1940a, 1940b; 3) Poly-S-carbobenzyl-L-cysteine Mowry, 1956, 1978) or cysteine-cystine (approx.mol. wt. 10,400) rich substances (Dempsey et al., 1950 ; 4) Poly-glycine Pearse, 1951; Lillie et al., 1954). In elastic (approx. mol. wt. 6,000) fibers, Cooper (1971) postulated that the 5) Poly-L-histidine induction of basophilia by permanganate (approx. mol. wt. 80,000) oxidation is a function of protein (tyrosine) 6) Poly-L-leucine rather than carbohydrate determinants. (approx. mol. wt. 15,000) The difference in post-oxidation basophilia 7) Poly-L- among specimens of different origins may (approx.wt. 30,000) be related to differences in the action of 8) Poly-L-tryptophan the oxidizing agents rather than the basic (approx.mol. wt. 5,000) dyes. 9) Poly-L-tyrosine In the present study, oxidation-induced (approx.mol wt. 130,000) basophilia was examined, with special reference to the effects of certain oxi- Dye solutions were prepared by dis- dizing agents widely used in histochemical solving 50 mg of azure A (Chroma, C. I. studies. No. 52005) or crystal violet (Merck, C.I. No. 42555) in 50 ml of 0.1 M HCI-sodium Materials and Methods citrate buffer (pH 1.2, 2.0, or 3.0). Other thiazines such as thionine, toluidine blue The oxidizing agents and periods of 0 and methylene blue were also used. oxidation are listed in Table 1. Specimens The deparaffinized tissue sections and from the thyroid gland, hypophysis, pan- model tissue sections were treated with creas, aorta, small intestine, stomach, one of the oxidizing agents listed in skin, and cerebellum of rabbits and guinea Table 1 for the stated periods. Sections pigs were obtained. The tissues were treated with a reagent containing KMnO4 Oxidation-Induced Basophilia of Various Tissues 371 were decolorized with 1% or conditions used and, moreover, completely 2-5% aqueous sodium . After eliminated the basophilia of intestinal each oxidative treatment, the sections goblet cell mucin, pancreatic acinar cells, were washed for 10 min under running Purkinje cells, and nuclei. At pH 2.0 and and then stained for 5 min with a 1.2, increased basophilia was not observed dye solution. As controls, adjacent serial in tissue sections subjected to oxidation sections were stained in the same manner with the different oxidizing agents, ex- without prior oxidation. cept for pilary cortex which exhibited To examine the effect of each of the slight basophilia. oxidizing agents on the chemical end groups of tissue sections, use was made II. Model tissue sections (Table 2) of the diazotization coupling reaction for The poly-L-tyrosine floccules in gelatin tyrosine (Lillie, 1957), the DDD reaction were stained with azure A (pH 3.0) or for the SH group of cysteine and the SS other basic dyes after KMnO4 or KMnO4 group of cystine (Barrnett and Seligman, •I-12SO4treatment (Fig . 13). After KMnO4, 1952), the rosindole reaction for trypto- KMnO4 H2SO4, performic acid or peracetic phan (Glenner, 1957), the Sakaguchi reac- acid treatment, the poly-S-benzyl-L-cy- tion for arginine (Baker, 1947), and the steine, poly-S-carbobenzyl-L-cysteine and Schiff reaction for . poly-L-tryptophan floccules were stained by azure A (pH 3.0) and other basic dyes Results (Figs. 14-19). Periodic acid exposure be- yond 8 hr induced increased basophilia in I. Tissue sections two synthetic polycysteine floccules (Fig. Table 1 shows the azure A (pH 3.0) 20). At pH 2.0 and 1.2, the basophilia of staining pattern of oxidation-treated tis- the model tissues was increased in the sue section. The staining pattern pro- same polypeptides as mentioned above duced by KMnO4 oxidation closely resem- for pH 3.0. bled that yielded by KMnO4' H2SO4. Thy- roidal colloid, hypophyseal neurosecretory III. Effect of oxidizing agents on histo- substances, aortic and dermal elastic fibers, chemical reactions (Tables 3 and 4) pancreatic acinar cells and B cells, pilary 1) KMnO4 and KMnO, • H2SO4 cortex, intestinal goblet cell mucin gastric The diazotization coupling, DDD and mucin, gastric chief cells and Purkinje rosindole reactivities of all tissues were cells were strongly stained with azure A found to be largely or completely de- or other basic dyes after KMnO4 or stroyed. The KMnO4•1-12SO4-Schiff reac- KMn04. H2SO4 treatment (Figs. 1-8). Per- tion was strong in thyroidal colloid, gas- formic and peracetic acid treatment tric mucin and pilary cortex, while the induced basophilia in hypophyseal neuro- KMn04-Schiff reaction was strong in secretory substances, pancreatic B cells pilary cortex. and pilary cortex (Figs. 9-11), but only 2) Performic acid and peracetic acid pilary cortex exhibited increased baso- The DDD and rosindole reactivities of philia after periodic acid treatment (Fig. all tissues were blocked. No effect on 12). Pancreatic B cells and hypophyseal the diazotization coupling or Sakaguchi neurosecretory substances were strongly reaction was noted. Only pilary cortex stained with basic dyes, especially in was positive for the Schiff reagent. Zenker-formol-fixed tissue sections. Chro- 3) Periodic acid mic acid proved ineffective under the The diazotization coupling, DDD and Table 1. Azure A (pi-I 3.0) staining reactions of various tissues after treatment with oxidizing agents

i) equal parts of 0.3% aqueous KMnO4 and 0.3% H2SO4, 5 min ; 2) 0.5% , 1 hr ; 8) see Ref. 30, 5-30 min ; 4) see Ref . 24, reagent was aged overnight before use, 2hr ; 1% aqueous solution, 2 hr at 60°C ; 6) 5% aqueous solution, 1-2 hr at 60°C. 0=no stain, ± questionable or weak stain, 1= definite positive stain, 2, 3, 4= progressively stronger staining Table 2. Azure A (pH 3.0) Staining reactions of model tissues after treatment with oxidizing agents')

C

0)

CD—• C-

0) U) 0

0'

C

(,* (,) C CD Cl2

I) for composition and oxidation periods , see legend to Table 1., 2) hydrochloride, type 1I-B, lot no. 37C-5008, 3) lot no. 95C-5018, 4) type I-C , lot no. 45C-5046, 5) type I, lot no. 1040-0256, 6) type 1-C, lot no. 980-5026, 7) type II-C, lot no. 105C-5022, 8) type 1-C, lot no. 113C-5390, 9) type II, lot no. 18C-5045, ") type I-B, lot no. 18C-5062 0=no stain, ± =questionable or weak stain, 1= definite positive stain, 2, 3, 4=progressively stronger staining , ) =oxidation for 8 hr

Coa c,0

Table 3. Effect of oxidizing agents on histochernical reactions for protein

vq.

a=control, b=--potassium permanganate-, c=potassium permanganate, d=performic acid, e=peracetic acid, f=periodic acid, g=chromic acid, ( ) =reaction of SS groups, O=no reaction, ±=questionable or weak reaction, 1=definite positive reaction, 2, 3, 4=progressively stronger reaction Table 4. Schiff reactions of various tissues after treatment with oxidizing agents

—• ca .

0 0 5"

Cr,

cri ss) (r) 0

0

ZA" Cl) (t) Cl)

Ono reaction, ± = questionable or weak reaction, 1= definite positive reaction, 2, 3, 4,=progressively stronger reaction 4,4CA) 376 M. Akita rosindole reactivities of all tissues were increase the slight basophilia of elastic blocked. Thyroidal colloid, gastric mucin, fibers, an observation that is in accord intestinal goblet cell mucin and gastric with the low sulfur content of elastin. chief cells were strongly positive for the Cooper (1971) noted that elastic fibers Schiff reagent. develop strong basophilia upon prolonged. 4) Chromic acid permanganate oxidation, and he postu- The histochemical reactivities except lated that the alkylphenolic tyrosine for the Sakaguchi reaction, of all tissues residue in elastic fibers is oxidized to were blocked. alkylcarboxylic acid via an alkylaldehyde intermediate stage. The tissue-staining Discussion pattern produced by potassium permanga- nate oxidation closely resembles that of According to Nicolet and Shinn (1939), the diazotization coupling reaction for periodic acid attacks tryptophan, methio- tyrosine and the DDD reaction for SS nine, cystine and a-hydroxyamino-acid and SH groups. The present findings on serine. Toennies and Homiller (1942) postoxidative histochemical reactions for showed that performic acid reacts only protein suggest that potassium perman- with tryptophan, cystine and methionine ganate attacks the groups of tyro- among the amino acids. Alexander et al. sine, the SH groups of cysteine and the (1950), who used KMnO4 in 0.5 N H2SO4, indole groups of tryptophan. In the model succeeded in oxidizing 30% of the cystine tissues, cysteine, tyrosine and tryptophan present in horn keratin to cysteic acid exhibited increasing basophilia with potas- and other products. These authors found sium permanganate. On the other hand, that if peracetic acid was used for the performic and peracetic acid did not oxidation, as much as 75% of the cystine attack the phenol groups of tyrosine. was oxidized in 2 hr, and cysteic acid was Furthermore, the tyrosine floccules of the considered to be the only reaction product. model tissues did not exhibit oxidation- Dempsey et al. (1950) reported increased induced basophilia. The specificity of basophilia of tissue proteins after oxida- potassium permanganate oxidation is tion with periodic acid, raising the possi- lower than that of performic and per- bility that oxidation products of cystine acetic acid oxidation. could be demonstrated by means of basic Pearse (1951) and Bonomi (1951) found dyes such as methylene blue. Cystine- that only performic acid and peracetic cysteine rich substances such as hypo- acid were satisfactory in practice : peri- physeal neurosecretory substances and odic acid, potassium permanganate, hy- pancreatic B cell granules, stain with droxy peroxide, and perchloric acid were many basic dyes aftcr oxidation with unsuitable for selective oxidation of SS potassium permanganate, performic acid groups. However, in the present model or peracetic acid (Adams and Sloper, 1955, tissues, performic and peracetic acid 1956 ; Bangle, 1954, 1956 ; Gabe, 1969). oxidation increased the basophilia of After potassium permanganate oxida- tryptophan floccules. These results indi- tion, tissue components such as pilary cate that at least the amino acids which cortex, which show a positive reaction increase basophilia after oxidation with for SS and SH groups, manifest increased these reagents, are not always exclusively basophilia. According to Dempsey and attributable to cystine (or cysteine). Lansing (1954), oxidation with reagents Periodic acid oxidation of tissue sec- such as periodic acid does not markedly tions resulted in only moderate basophilia Oxidation-Induced Basophilia of Various Tissues 377 of pilary cortex, as compared to the basophilia in all tissues. This suggests intense reaction obtained with performic that chromic acid, under the present and peracetic acid oxidation. The cysteine conditions, further attacks and destroys floccules of model tissues were stained , probably converting them first upon prolonging the period of oxidation. to carboxyl groups and then to carbon Lillie and Fullmer (1976) noted that 18-hr dioxide. fixation at 24°C in 1% periodic acid-10% formol did not induce basophilia of kera- References tin or other structures, although the usual structures were rendered Schiff- 1) Adams, C.W.M. and Sloper, J.C. : Tech- nique for demonstrating neurosecretory positive. The oxidation effect of periodic material in the human hypothalamus. acid on SS and SH groups was inferior Lancet 6865 : 651-652, 1955. to that of performic and peracetic acid. 2) Adams, C.W.M. and Sloper, J.C. : The According to Lillie et al. (1954), chro- hypothalamic elaboration of posterior mic acid (5%, 30 and 60 min at 60°C) pituitary principles in man, the rat and largely or completely destroyed nuclear dog ; histochemical evidence derived from basophilia, and Fisher (1953) reported that a performic acid-alcian blue reaction for chromic acid (5%, 30 min at 25-30°C) cysteine. J. Endocrinol. 13 : 221-228, 1956. blocked the basophilia of thyroidal colloid. 3) Alexander, P., Hudson, R.F. and Fox, In the present study, the basophilia of all M. : The reaction of oxidizing agents tested tissues was abolished by chromic with wool. 1. The division of cystine into two fractions of widely differing reactivi- acid oxidation. Furthermore, chromic acid ties. Biochem. J. 46 : 27-32, 1950. blocked the reactivity of all histochemical 4) Baker, J.R. : The histochemical recogni- methods except that of the Sakaguchi tion of certain guanidine derivatives. reaction. The oxidation effect of chromic Quart. J. Micr. Sci. 88: 115-121, 1947. acid is much stronger than that of other 5) Bangle, R.: Gomori's paraldehyde-fuchsin oxidizing agents, probably because chro- stain. I. Physicochemical and staining mic acid destroys acidic groups, such as properties of the dye. J. Histochem. the phosphoric groups of nucleic acids Cytochem. 2: 291-299, 1954. and sulfate or the carboxylic groups of 6) Bangle, R.: Factors influencing the stain- mucosubstances. ing of beta-cell granules in pancreatic As pointed out by Bignardi (1939, 1940a, islets with various basic dyes, including 1940b) and Mowry (1956, 1978), carbo- paraldehyde-fuchsin. Am. J. Pathol. 32: 346-362, 1956. hydrates also exhibit basophilia after 7) Barrnett, R.J. and Seligman, A.M. : Histo- oxidation. The generally weaker staining chemical demonstration of protein-bound seen with potassium permanganate-Schiff sulfhydryl groups. Science 116: 323-327, or chromic acid-Schiff, as compared to 1952. the PAS reaction, has been attributed to 8) Bignardi, C.: Ricerche prelimnari sulla the fact that potassium permanganate istochimica del tessuto cartilagineo. Atti and chromic acid go on to oxidize alde- Soc. Natur. Matem. Modena. 70: 97-102, hydes to (Lillie, 1951). It 1939. appears likely that the PAS-positive sites 9) Bignardi, C.: Cellule mucose e cellule mucoidi. IV. Esterificazione solforica della exhibit basophilia because of the car- sostanza mucoide e sua dimonstrazion boxylic acid produced by oxidation with istochimica. Atti Soc. Natur. Matem. potassium permanganate or chromic acid. Modena. 71 : 59-61, 1940a. However, in the present study, chromic 10) Bignardi, C.: Sulla probabile presenza acid (5%, 2 hr at 60°C) did not induce di un polisaccharide non esterificato nelle 378 M. Akita

cellule mucose. Boll. Soc. Ital. Biol. Sper. 22) Lillie, R.D. : Adaptation of the Morel 15 : 593-594, 1940b. Sisley protein diazotization procedure to 11) Bonomi, U. : Sul comportamento tinto- the histochemical demonstration of pro- riale delle B-cellule delle isole pancrea- tein bound tyrosine. J. Histohem. Cyto- tiche. Boll. Soc. Ital. Biol. Sper. 21: chem. 5 : 528-532, 1957. 1418-1420, 1951. 23) Lillie, R,D., Bangle, R. and Fisher, E.R. 12) Cooper, J.H. : The Casella reaction and Metachromatic basophilia of keratin after the permanganate-induced basophilia of oxidation-cleavage disulfide bonds. J. human elastica. J. Histochem. Cytochem. Histochem. Cytochem. 2 : 95-102, 1954. 19: 564-568, 1971. 24) Lillie, R.D. and Fullmer, H.M. : Histo- 13) Dempsey, E.W. and Lansing, A.I. : Elastic pathologic Technic and Practical Histo- tissue. Int. Rev. Cytol. 3: 437-453, 1954. chemistry, 4th Ed., McGraw-Hill, Inc., 14) Dempsey, E.W., Singer, M. and Wislocki, New York, 1976, p. 220. G.B. : The increased basophilia of tissue 25) Ibid., p. 230. proteins after oxidation with periodic 26) Mowry, R.W.: Improved method for the acid. Stain Technol. 25: 73-80, 1950. induction of metachromasia induced by 15) Fisher, E.R. Observations on thyroid sulfation. J. Histochem. Cytochem. 4: colloid. Arch. Pathol. 56: 275-285, 1953. 409-410, 1956. 16) Gabe, M.: Action du pH de la solution 27) Mowry, R.W.: Aldehyde fuchsin stain- colorante sur la basophilie apparue dans ing, direct or after oxidation : problems des produits de secretion riches en cystine and remedies, with special reference to apr6s certaines oxydations. C.R. Acad. human pancreatic B cells, pituitaries, and Sci. (Paris) 268: 1518-1520, 1969. elastic fibers. Stain Technol, 53 : 141-154, 17) Gabe, M.: Histological Techniques, Mas- 1978. son, Paris, 1976, p. 335. 28) Nicolet, B.H. and Shinn, L.A. : The action 18) Glenner, G.G. : The histochemical dem- of periodic acid on a-amino . J. onstration of indole derivatives by the Am. Chem. Soc. 61 : 1615, 1939. rosindole reaction of E. Fischer. J. Histo- 29) Pearse, A.G.E.: The histochemical dem- chem. Cytochem. 5: 297-304, 1957. onstration of keratin by methods involv- 19) Harada, K.: Rapid demonstration of ing selective oxidation. Quart. J. Micr. juxtaglomerular granules with alcoholic Sci. 92 : 393-402, 1951. crystal violet. Stain Technol. 45: 71-74, 30) Pearse, A.G.E.: Histochemistry Theore- 1970. tical and Applied, Vol. 1, 3rd Ed., J. & 20) Kramer, H. and Windrum, G.M. : Sulfa- A. Churchill, Ltd., London, 1975, p. 701. tion techniques in histochemistry with 31) Smith, C.L. : Rapid demonstration of jux- special reference to metachromasia. J. taglomerular granules in mammals and Histochem. Cytochem. 2: 196-208, 1954. birds. Stain Technol. 41 : 291-294, 1966. 21) Lillie, R.D.: Histochemical comparison 32) Toennies, G. and Homiller, R.P. : The of the Casella, Bauer, and periodic acid oxidation of amino acids by hydrogen oxidation-Schiff leucofuchsin technics. peroxide in . J. Am. Chem. Stain Technol. 26 : 123-136, 1951. Soc. 64 : 3054-3056, 1942. Oxidation-Induced Basophilia of Various Tissues 379

PLATES 380 M. Akita

Explanation of Figures

Plate I

Figs. 1-6. Azure A (pH 3.0) staining reaction of tissue sections after KMnO4-H2SO4 treatment. Fixation was by 10% formol (Figs. 1, 2 and 4-6) or Zenker-formol (Fig. 3).

Fig. 1. Rabbit thyroid gland. x 300

Fig. 2. Rabbit hypophysis. x 300

Fig. 3. Rabbit pancreas. x 300

Fig. 4. Rabbit aorta. x 300

Fig. 5. Rabbit small intestine. x 300

Fig. 6. Rabbit stomach. x 300 381

Plate I

M. Akita 382 M. Akita

Plate II

Figs. 7-12. Azure A (pH 3.0) staining reaction of tissue sections after treatment with KMnO4 -142SO4 (Figs . 7 and 8), performic acid (Figs. 9-11) or periodic acid (Fig. 12). Fixation was by 10% formol (Figs. 7-9, 11 and 12) or Zenker-formol (Fig. 10).

Fig. 7. Rabbit skin. x 300

Fig. 8. Robbit cerebellum. x 300

Fig. 9. Rabbit hypophysis. x 300

Fig. 10. Rabbit pancreas. x 300

Fig. 11. Rabbit skin. x 300

Fig. 12. Rabbit skin. x 300 383

Plate II

M. Akita 384 M. Akita

Plate III

Figs. 13-20. Azure A (pH 3.0) staining reaction of model tissue sections after oxidation with KMn04. H2SO4 (Figs. 13-16) , performic acid (Figs. 17-19) or periodic acid (Fig. 20) . The oxidation with periodic acid was for 8 hr.

Fig. 13. Poly-L-tyrosine. x 300

Fig. 14. Poly-S-carbobenzyl-L-cysteine. x 300

Fig. 15. Poly-S-benzyl-L-cysteine. x 300

Fig. 16. Poly-L-tryptophan. x 300

Fig. 17. Poly-S-carbobenzyl-L-cysteine. x 300

Fig. 18. Poly-S-benzyl-L-cysteine. x 300

Fig. 19. Poly-L-tryptophan. x 300

Fig. 20. Poly-S-carbobenzyl-L-cysteine. x 300 385

Plate Ill

M. Akita